Compositions and methods for synthesis of phosphorylated molecules

ABSTRACT

The invention provides compositions and methods for synthesis of phosphorylated organic compounds, including nucleoside triphosphates.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/652,475, filed Apr. 4, 2018 which is hereby incorporated by referenceherein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No.N66001-16-2-4061, awarded by the Defense Advanced Research ProjectsAgency (DARPA). The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Polymerases are among the most powerful tools in the molecular biologyarsenal, permitting researchers to precisely synthesize the DNAsequences of genes, gene clusters, and entire genomes for diverseapplications ranging from basic research to biotechnology and medicine(Loeb et al., 2008, Nat. Rev. Genet. 9, 594-604; Wu et al., 2017, Nat.Rev. Chem. 1, 0068). Polymerase-mediated DNA synthesis has reduced thecost of DNA sequencing by allowing next-generation sequencing (NGS)platforms to read massive numbers of amplicons and has enabled digitalarchiving by compressing information into strands of DNA sequences thatcan be read by NGS analysis and decoded by bioinformatic assembly(Goodwin et al., 2016, Nat. Rev. Genet. 17, 333-351; Erlich andZielinski, 2017, Science 355, 950-954). Engineered polymerases,developed by directed evolution, have also grown in demand by enablingthe synthesis of artificial genetic polymers with backbone structuresthat are distinct from those found in nature (Chen and Romesberg, 2014,FEBS Lett. 588, 219-229; Loakes and Holliger, 2009, Chem. Commun.,4619-4631). Such enzymes are now used to support the evolution ofaffinity reagents (aptamers) and catalysts with molecular compositionsthat are better equipped to function in harsh biological environments(Pinheiro et al., 2012, Science 336, 341-344; Yu et al., 2012, Nat.Chem. 4, 183-187; Taylor et al., 2015, Nature 518, 427-430; Wang et al.,2018, Nat. Commun., in press).

Today, no single strategy has been developed that can be applieduniversally to the synthesis of all nucleoside triphosphates. NaturalDNA triphosphates (dNTPs) used to support traditional DNA synthesisapplications are manufactured using enzymatic methods, while modifiednucleoside triphosphates developed for biotechnology and medicinalpurposes are produced by chemical synthesis (Burgess and Cook, 2000,Chem. Rev. 100, 2047-2060; Kore and Srinivasan, 2013, Curr. Org. Syn.10, 903-934; Hollenstein, 2012, Molecules 17, 13569-13591). In general,enzymatic strategies suffer from high substrate specificity (lowtolerance for analogs), while synthetic routes struggle with functionalgroup compatibility, difficult reaction conditions, and yield.

Even more problematic is the requirement for both strategies to separatehighly polar nucleoside triphosphates from polar side products usinghigh performance liquid chromatography (HPLC). In academic environments,HPLC purification is a tedious process that limits the scale ofnucleoside triphosphate synthesis to a few tens of milligrams ofcompound per week and requires subsequent freeze-drying steps that canlead to compound degradation.

Efforts to synthesize nucleoside triphosphates independent of HPLCpurification first appeared in 2014 with a phosphorylation strategy thatinvolves iterative cycles of coupling, oxidation, and deprotection(Cremosnik et al., 2014, Angew. Chem. Int. Ed. 53, 286-289; Hofer et al,2015, Chemistry 21, 10116-10122; Sau and Chaput, 2017, Org. Lett. 19,4379-4382). This is a P(III)-based reagent method that relies onderivatized phosphoramidite reagents to facilitate P—O bond formation.Free nucleosides are converted to nucleoside triphosphates through theproduction of P(III)-P(V) anhydride intermediates that are oxidized anddeprotected during each cycle of P(V) phosphate generation (Cremosnik etal., 2014, Angew. Chem. Int. Ed. 53, 286-289). The iterative strategybenefits from high yields, rapid coupling rates, and convenient(non-dry) reaction conditions but is marked by several disadvantagesthat include: (1) the need for quantitative functional grouptransformations, which are necessary to avoid the accumulation ofunwanted side products; (2) prolonged deprotection conditions due toslow removal of the second P—O protecting group on the phosphorylatingreagent; (3) incompatibility with certain chemical reagents andnucleoside protecting groups; and (4) a lengthy pathway that involves 9functional group transformations to convert each nucleoside to itscorresponding triphosphate (Cremosnik et al., 2014, Angew. Chem. Int.Ed. 53, 286-289; Hofer et al, 2015, Chemistry 21, 10116-10122; Sau andChaput, 2017, Org. Lett. 19, 4379-4382).

Thus, there is a need in the art for novel compositions and methods forsynthesis of phosphorylated molecules. The present invention satisfiesthis unmet need.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to a composition comprising anorganic molecule comprising a phosphate moiety of formula (1):

wherein:

m is an integer from 0 to 5;

n is an integer from 0 to 5;

X is O or CH₂; R₁₁ is an aryl, a heteroaryl, an alkyl, a cycloalkyl, analkenyl, an alkynyl, an alkyl-aryl, an aryl-alkyl, or a silyl group; R₁₂is hydrogen, null, a substituted tetahydrofuranyl group, or analkyl-substituted tetahydrofuranyl group; and

each occurrence of R₁₃ is independently hydrogen or null.

In one embodiment, R₁₁ is substituted. In one embodiment, R₁₁ issubstituted with an alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heteroaryl, halogen, —CN, —OR₁₄, or —N(R₁₄)₂ group, wherein eachoccurrence of R₁₄ is independently selected from hydrogen, alkyl,alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, and halogen.

In one embodiment, R₁₁ is

In one embodiment, the compound of formula (1) is a compound of formula(1a):

wherein:

R_(1a) is an aryl, a heteroaryl, an alkyl, a cycloalkyl, an alkenyl, analkynyl, an alkyl-aryl, or an aryl-alkyl. In one embodiment, R_(1a) issubstituted. In one embodiment, R_(1a) is substituted with an alkyl,alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, halogen, —CN, —OR_(11a),or —N(R_(11a))₂ group, wherein each occurrence of R_(11a) isindependently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl,cycloalkyl, heteroaryl, and halogen.

In one embodiment, R_(1a) is:

In one embodiment, the compound of formula (1a) is:

In one embodiment, the compound of formula (1) is a compound of formula(1b):

wherein:

R_(1b) is a hydroxy protecting group.

In one embodiment, R_(1b) is an aryl, a heteroaryl, an alkyl, acycloalkyl, an alkenyl, an alkynyl, an alkyl-aryl, or an aryl-alkyl. Inone embodiment, R_(1b) is substituted.

In one embodiment, R_(1b) is substituted with an alkyl, alkenyl,alkynyl, aryl, cycloalkyl, heteroaryl, halogen, —CN, —OR_(11b), or—N(R_(11b))₂ group, wherein each occurrence of R_(11b) is independentlyselected from hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heteroaryl, or halogen.

In one embodiment, R_(1b) is

In one embodiment, the compound of formula (1b) is:

In one embodiment, the compound of formula (1) is a compound of formula(1c):

wherein:

m is an integer from 0 to 5;

n is an integer from 0 to 5;

X is O or CH₂;

R_(11c) is an aryl, a heteroaryl, an alkyl, a cycloalkyl, an alkenyl, analkynyl, an alkyl-aryl, an aryl-alkyl, or a silyl group;

each occurrence of R_(13c) is independently hydrogen or null;

R_(15c) is hydrogen, aryl, or heteroaryl; and

R_(16c) is a nitrogenous base, wherein the nitrogenous base is a naturalnitrogenous base or artificial nitrogenous base.

In one embodiment, R_(11c) is

In one embodiment, R_(15c) is phenyl.

In one embodiment the compound of formula (1c) is:

In one embodiment, the compound of formula (1) is a compound of formula(1d):

wherein:

m is an integer from 0 to 5;

n is an integer from 0 to 5;

X is O or CH₂;

R_(11d) is an aryl, a heteroaryl, an alkyl, a cycloalkyl, an alkenyl, analkynyl, an alkyl-aryl, an aryl-alkyl, or a silyl group;

each occurrence of R_(13d) is independently hydrogen or null;

R_(15d) is hydrogen, aryl, or heteroaryl; and

R_(16d) is a nitrogenous base, wherein the nitrogenous base is a naturalnitrogenous base or artificial nitrogenous base.

In one embodiment, R_(11d) is

In one embodiment, R_(15d) is phenyl.

In one embodiment, the compound of formula (1d) is:

In one embodiment, the compound of formula (1) is a compound of formula(β):

wherein:

m is an integer from 0 to 5;

n is an integer from 0 to 5;

X is O or CH₂;

R_(11c) is an aryl, a heteroaryl, an alkyl, a cycloalkyl, an alkenyl, analkynyl, an alkyl-aryl, an aryl-alkyl, or a silyl group;

each occurrence of R_(13e) is independently hydrogen or null;

R_(15e) is hydrogen, aryl, acetyl, or heteroaryl; and

R_(16e) is a nitrogenous base, wherein the nitrogenous base is a naturalnitrogenous base or artificial nitrogenous base.

In one embodiment, R_(11c) is

In one embodiment, R_(15e) is phenyl.

In one embodiment, the compound of formula (1e) is:

In one embodiment, the invention relates to a compound represented byformula (2):

wherein n is an integer from 1 to 10, and wherein R₂ is a support.

In one embodiment, the invention relates to a compound represented byformula (3):

wherein p is an integer from 0 to 10, and R₃ is a support.

In one embodiment, the invention relates to a compound represented byformula (4):

wherein q is an integer from 0 to 10, and

r is an integer from 0 to 10. In one embodiment, q is 1. In oneembodiment, r is 1.

In one embodiment, the compound of formula (4) is

In one embodiment, the invention relates to a method of synthesizing anucleoside (n)phosphate, wherein (n) is: di, tri, tetra, penta, or hexa,comprising a) contacting an activated nucleoside monophosphate with acompound represented by formula (1), a compound represented by formula(1a), a compound represented by formula (1b), a compound represented byformula (1a), a compound represented by formula (2), a compoundrepresented by formula (3) or a compound represented by formula (4), andan acid catalyst to catalyze the formation of a reaction intermediate,and b) contacting the reaction intermediate with a base to induceformation of a nucleoside (n)phosphate.

In one embodiment, the acid catalyst is ZnCl₂.

In one embodiment, the activated nucleoside monophosphate is representedby formula (5)

wherein R₅ is hydrogen, aryl, or heteroaryl; and R₆ is a nitrogenousbase, wherein the nitrogenous base is a natural nitrogenous base orartificial nitrogenous base.

In one embodiment, the activated nucleoside monophosphate is anactivated monophosphate of adenosine, cytidine, guanosine, uridine,2′-deoxyadenosine, 2′-deoxycytidine, 2′-deoxyguanosine, thymidine,inosine, 9-(β-D-arabinofuranosyl)adenine,1-(β-D-arabinofuranosyl)cytosine, 9-(β-D-arabinofuranosyl)guanine,1-(β-D-arabinofuranosyl)uracil, 9-(β-D-arabinofuranosyl)hypoxanthine,1-(β-D-arabinofuranosyl)thymine, 3′-azido-3′-deoxythymidine,3′-azido-2′, 3′-dideoxyuridine, 3′-azido-2′, 3′-dideoxycytidine,3′-azido-2′, 3′-dideoxyadenosine, 3′-azido-2′, 3′-dideoxyguanosine,3′-azido-2′, 3′-dideoxyinosine, 3′-deoxythymidine, 2′,3′-dideoxyuridine, 2′, 3′-dideoxyinosine, 2′, 3′-dideoxyadenosine, 2′,3′-dideoxycytidine, 2′, 3′-dideoxyguanosine, 9-(2,3-dideoxy-1-β-D-ribofuranosyl)-2, 6-diaminopurine, 3′-deoxy-2′,3′-didehydrothymidine, 2′, 3′-didehydro-2′, 3′-dideoxyuridine, 2′,3′-didehydro-2′, 3′-dideoxycytidine, 2′, 3′-didehydro-2′,3′-dideoxyadenosine, 2′, 3′-didehydro-2′, 3′-dideoxyguanosine, 2′,3′-didehydro-2′, 3′-dideoxyinosine, 3-deazaadenosine, 3-deazaguanosine,3-deazainosine, 7-deazaadenosine, 7-deazaguanosine, 7-deazainosine,6-azauridine, 6-azathymidine, 6-azacytidine, 5-azacytidine,9-(β-D-ribofuranosyl)-6-thiopurine,6-methylthio-9-(β-D-ribofuranosyl)purine,2-amino-9-(β-D-ribofuranosyl)-6-thiopurine,2-amino-6-methylthio-9-(β-D-ribofuranosyl)purine, 5-fluorocytidine,5-iodocytidine, 5-bromocytidine, 5-chlorocytidine, 5-fluorouridine,5-iodouridine, 5-bromouridine, 5-chlorouridine, 2′-C-methyladenosine,2′-C-methylcytidine, 2′-C-methylguanosine, 2′-C-methylinosine,2′-C-methyluridine, 2′-C-methylthymidine, 2′-deoxy-2′-fluoroadenosine,2′-deoxy-2′-fluorocytidine, 2′-deoxy-2′-fluoroguanosine,2′-deoxy-2′-fluorouridine, 2′-deoxy-2′-fluoroinosine,2′-α-fluorothymidine, 2′-deoxy-2′-fluoroarabinoadenosine,2′-deoxy-2′-fluoroarabinocytidine, 2′-deoxy-2′-fluoroarabinoguanosine,2′-deoxy-2′-fluoroarabinouridine, 2′-deoxy-2′-fluoroarabinoinosine,2′-3-fluorothymidine, 2′-O-methyladenosine, 2′-O-methylcytidine,2′-O-methylguanosine, 2′-O-methylinosine, 2′-O-5-dimethyluridine,2′-C-ethynylcytidine, 2′-C-ethynylguanosine, 2′-C-ethynyluridine,2′-C-ethynylinosine, 2′-C-ethynyl-5-methyluridine,3′-C-ethynyladenosine, 3′-C-ethynylcytidine, 3′-C-ethynylguanosine,3′-C-ethynyluridine, 3′-C-ethynylinosine, 3′-C-ethynyl-5-methyluridine,3′-deoxyadenosine, 3′-deoxycytidine, 3′-deoxyguanosine, 3′-deoxyuridine,3′-deoxyinosine, 4′-C-ethynyladenosine, 4′-C-ethynylcytidine,4′-C-ethynylguanosine, 4′-C-ethynyluridine, 4′-C-ethynylinosine,4′-C-ethynylthymidine, 4′-C-methyladenosine, 4′-C-methylcytidine,4′-C-methylguanosine, 4′-C-methyluridine, 4′-C-methylinosine,4′-C-methylthymidine, 2′-C-methyl-7-deazaadenosine,2′-C-methyl-7-deazaguanosine, 2′-C-methyl-3-deazaadenosine,2′-C-methyl-3-deazaguanosine, 2′-O-methyl-7-deazaadenosine,2′-O-methyl-7-deazaguanosine, 2′-O-methyl-3-deazaadenosine,2′-O-methyl-3-deazaguanosine, 2′-C-methyl-6-azauridine,2′-C-methyl-5-fluorouridine, 2′-C-methyl-5-fluorocytidine,2′-C-methyl-2-chloroadenosine, 2′-deoxy-7-deazaadenosine,2′-deoxy-3-deazaadenosine, 2′-deoxy-7-deazaguanosine,2′-deoxy-3-deazaguanosine, 2′-deoxy-6-azauridine,2′-deoxy-5-fluorouridine, 2′-deoxy-5-fluorocytidine,2′-deoxy-5-iodouridine, 2′-deoxy-5-iodocytidine,2′-deoxy-2-chloroadenosine, 2′-deoxy-2-fluoroadenosine,3′-deoxy-7-deazaadenosine, 3′-deoxy-7-deazaguanosine,3′-deoxy-3-deazaadenosine, 3′-deoxy-3-deazaguanosine,3′-deoxy-6-azauridine, 3′-deoxy-5-fluorouridine, 3′-deoxy-5-iodouridine,3′-deoxy-5-fluorocytidine, 3′-deoxy-2-chloroadenosine, 2′,3′-dideoxy-7-deazaadenosine, 2′, 3′-dideoxy-7-deazaguanosine, 2′,3′-dideoxy-3-deazaadenosine, 2′, 3′-dideoxy-3-deazaguanosine, 2′,3′-dideoxy-6-azauridine, 2′, 3′-dideoxy-5-fluorouridine, 2′,3′-dideoxy-5-fluorouridine, 2′, 3′-dideoxy-5-iodocytidine, 2′,3′-dideoxy-2-chloroadenosine, 2′, 3′-dideoxy-β-L-cytidine, 2′,3′-dideoxy-β-L-adenosine, 2′, 3′-dideoxy-β-L-guanosine,3′-deoxy-β-L-thymidine, 2′, 3′-dideoxy-5-fluoro-β-L-cytidine,3-L-thymidine, 2′-deoxy-β-L-cytidine, 2′-deoxy-β-L-adenosine,2′-deoxy-β-L-guanosine, 2′-deoxy-β-L-inosine, β-L-cytidine,β-L-adenosine, 3-L-guano sine, β-L-uridine, β-L-inosine, 2′,3′-didehydro-2′, 3′-dideoxy-β-L-cytidine, 2′,3′-didehydro-3′-dideoxy-β-L-thymidine, 2′, 3′-didehydro-2′,3′-dideoxy-β-L-adenosine, 2′, 3′-didehydro-2′, 3′-dideoxy-β-L-guanosine,2′, 3′-didehydro-2′, 3′-dideoxy-3-L-5-fluorocytidine, 2′-deoxy-2′,2′-difluorocytidine, 9-(β-D-arabinofuranosyl)-2-fluoroadenine,2′-deoxy-2′(E)-fluoromethylenecytidine, 2′-deoxy-2′(Z)-fluoromethylenecytidine, (−)-2′, 3′-dideoxy-3′-thiacytidine, (+)-2′,3′-dideoxy-3′-thiacytidine, 1-β-D-ribofuranosyl-1, 2,4-triazole-3-carboxamide, 1-β-L-ribofuranosyl-1, 2,4-triazole-3-carboxamide, 1-β-D-ribofuranosyl-1, 3-imidazolium-5-olate,1-β-L-ribofuranosyl-1, 3-imidazolium-5-olate,1-β-D-ribofuranosyl-5-ethynylimidazole-4-carboxamide,1-β-L-ribofuranosyl-5-ethynylimidazole-4-carboxamide,1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodouracil,1-(2-deoxy-2-fluoro-3-D-arabinofuranosyl)-5-iodocytosine,1-(2-deoxy-2-fluoro-3-L-arabinofuranosyl)-5-methyluracil,1-β-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil,E-5-(2-bromovinyl)-2′-deoxyuridine, 5-trifluoromethylthymidine,1-β-D-arabinoifuranosyl-5-propynyluracil,1-(2-deoxy-2-fluoro-1-β-D-arabinofuranosyl)-5-ethyluracil, 2′,3′-dideoxy-3′-fluoroguanosine, 3′-deoxy-3′-fluorothymidine, (+)-(1α, 2β,3α)-9-[2, 3-bis(hydroxymethyl)-1-cyclobutyl]adenine, (+)-(1α, 2β,3α)-9-[2, 3-bis(hydroxymethyl)-1-cyclobutyl]guanine, (+)-(1β, 2α,3β)-9-[2, 3-bis(hydroxymethyl)-1-cyclobutyl]guanine, (+)-(1β, 2α,3β)-9-[2, 3-bis(hydroxymethyl)-1-cyclobutyl]adenine, (1R, 3S,4R)-9-(3-hydroxy-4-hydroxymethylcyclopent-1-yl)guanine, (1 S, 2R,4R)-9-(1-hydroxy-2-hydroxymethylcyclopent-4-yl)guanine, (2R,4R)-9-(2-hydroxymethyl-1, 3-dioxolan-4-yl)-2, 6-diaminopurine, (2R,4R)-1-(2-hydroxymethyl-1, 3-dioxolan-4-yl)cytosine, (2R,4R)-9-(2-hydroxymethyl-1, 3-dioxolan-4-yl)guanine, (2R,4R)-1-(2-hydroxymethyl-1, 3-dioxolan-4-yl)-5-fluorocytosine, (1R, 2S,4S)-9-(4-hydroxy-3-hydroxymethyl-2-methylenecyclopent-4-yl]guanine, or(1 S, 3R,4S)-9-(3-hydroxy-4-hydroxymethyl-5-methylenecyclopent-1-yl]guanine.

In one embodiment, the invention relates to a composition comprising atleast one nucleoside (n)phosphate synthesized according to the method ofa) contacting an activated nucleoside monophosphate with a compoundrepresented by formula (1), a compound represented by formula (1a), acompound represented by formula (1b), a compound represented by formula(1a), a compound represented by formula (2), a compound represented byformula (3) or a compound represented by formula (4), and an acidcatalyst to catalyze the formation of a reaction intermediate, and b)contacting the reaction intermediate with a base to induce formation ofa nucleoside (n)phosphate.

In one embodiment, the composition comprises at least four nucleosidetriphosphates. In one embodiment, at least four nucleoside triphosphatesare deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate(dGTP), deoxycytidine triphosphate (dCTP), and deoxythymidinetriphosphate (dTTP).

In one embodiment, the composition is a pharmaceutical composition.

In one embodiment, the invention relates to a method of treating adisease of disorder in a subject in need thereof, the method comprisingadministering to the subject a pharmaceutical composition comprising atleast one nucleoside (n)phosphate synthesized according to the method ofa) contacting an activated nucleoside monophosphate with a compoundrepresented by formula (1), a compound represented by formula (1a), acompound represented by formula (1b), a compound represented by formula(1a), a compound represented by formula (2), a compound represented byformula (3) or a compound represented by formula (4), and an acidcatalyst to catalyze the formation of a reaction intermediate, and b)contacting the reaction intermediate with a base to induce formation ofa nucleoside (n)phosphate.

In one embodiment, the invention relates to a kit comprising at leastone nucleoside (n)phosphate synthesized according to the method of a)contacting an activated nucleoside monophosphate with a compoundrepresented by formula (1), a compound represented by formula (1a), acompound represented by formula (1b), a compound represented by formula(1a), a compound represented by formula (2), a compound represented byformula (3) or a compound represented by formula (4), and an acidcatalyst to catalyze the formation of a reaction intermediate, and b)contacting the reaction intermediate with a base to induce formation ofa nucleoside (n)phosphate.

In one embodiment, the composition comprises at least four nucleosidetriphosphates. In one embodiment, at least four nucleoside triphosphatesare deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate(dGTP), deoxycytidine triphosphate (dCTP), and deoxythymidinetriphosphate (dTTP).

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1 depicts exemplary structures of organic pyrophosphates, thephosphorylation reagent used in the synthesis of natural and modifiednucleoside triphosphates.

FIG. 2A through FIG. 2C, depicts a model for the synthesis of naturaland modified nucleoside triphosphates. FIG. 2A depicts the chemicalstructure of the p-toluene pyrophosphate reagent (1). FIG. 2B depicts adiagram of the synthesis of 5′-thymidine triphosphate from the reactionof activated thymidine monophosphate with the p-toluene pyrophosphatereagent of FIG. 2A. FIG. 2C depicts the chemical structure of anintermediate formed following step 1 of the reaction of FIG. 2B.

FIG. 3 depicts the chemical structures of an exemplary activatedmonophosphate (left) and monophosphate (right).

FIG. 4 depicts a model for the formation of dinucleoside diphosphate, aminor contaminant of the reaction of FIG. 2B.

FIG. 5 depicts chemical structure of pyrene pyrophosphate (left) andp-toluene pyrophosphate (right).

FIG. 6 depicts the chemical structures of exemplary nucleosidetriphosphates (from left to right: 5′-thymidine triphosphate (5′-TTP),3′-thymidine triphosphate (3′-TTP), and a-L-threofuranosyl 3′-thymidinetriphosphate (tTTP)) which have been synthesized using the methods ofthe invention.

FIG. 7 depicts the chemical structures of four naturally occurringnucleoside triphosphates which have been synthesized using the methodsof the invention.

FIG. 8 depicts a diagram of the synthesis of p-toluene pyrophosphatereagent.

FIG. 9 depicts a diagram of the synthesis of pyrene pyrophosphatereagent.

FIG. 10 depicts a diagram of the synthesis of activated thymidinemonophosphate.

FIG. 11 depicts HPLC analysis of a-L-threofuranosyl adenosinetriphosphate using the traditional Yoshikawa or Ludwig-Eckstein methods.Arrows indicate the location of the desired peak in the crude reactionmixture.

FIG. 12 depicts a diagram of the strategy and methodology development.Shown is a diagram for the scalable synthesis of natural and modifiednucleoside triphosphates using a new class of organopyrophosphatereagents. R, the aromatic substitution attached to a cleavablesulfonylethyl linker. LG, leaving group moiety. B, nucleobase moieties:adenine (A), guanine (G), cytosine (C), thymine (T).

FIG. 13 depicts the organic pyrophosphate reagent as verified by thecrystal structure.

FIG. 14A through FIG. 14C depicts a synthesis and purity comparison ofthymidine triphosphate. FIG. 14A depicts an application of theorganopyrophosphate reagent to the synthesis of naturalthymidine-5′-triphosphate. FIG. 14B depicts HPLC traces provided for theactivated monophosphate, fully protected triphosphate intermediate aftersilica gel purification, and thymidine triphosphate after precipitationas the sodium salt. FIG. 14C depicts a comparison of chemicallysynthesized thymidine 3′-triphosphate (dTTP) with a commercial standard.HPLC gradients are provided in the supplementary information.

FIG. 15 depicts synthesis of thymidine triphosphate analogs. Theorganopyrophosphate reagent was applied to the synthesis of thymidine3′-triphosphate, a-L-threofuranosyl thymidine 3′-triphosphate, andL-thymidine 5′-triphosphate. HPLC traces are provided afterprecipitation as the sodium salt.

FIG. 16A and FIG. 16B depict polymerase activity assay of chemicallysynthesized DNA and TNA nucleoside triphosphates. All four DNA and TNAnucleoside triphosphates were constructed using the organicpyrophosphate method. (a) Chemically synthesized dNTPs were evaluatedagainst commercial reagents in a standard PCR reaction. (b) Chemicallysynthesized tNTPs purified by silica gel chromatography (SG) wereevaluated against HPLC purified tNTPs (HPLC) in a polymerase-mediatedprimer-extension reaction using Kod-RI TNA polymerase.

FIG. 17 depicts a synthesis scheme for1-(2-(pyrenesulfonyl)ethyl)pyrophosphate (compound 7).

FIG. 18 depicts an ¹H NMR spectrum of synthesized 2-(Pyrenethio)ethanol(compound 2).

FIG. 19 depicts an ¹H NMR spectrum of synthesized2-(Pyrenesulfonyl)ethanol (compound 3).

FIG. 20 depicts a ¹³C NMR spectrum of synthesized compound 3.

FIG. 21 depicts a ¹³C NMR spectrum of synthesizedDibenzyl-1-(2-(pyrenesulfonyl)ethyl) monophosphate (compound 4).

FIG. 22 depicts an ³¹P NMR spectrum of synthesized compound 4.

FIG. 23 depicts an ¹H NMR spectrum of synthesized1-(2-(Pyrenesulfonyl)ethyl) monophosphate (compound 5).

FIG. 24 depicts a ¹³C NMR spectrum of synthesized compound 5.

FIG. 25 depicts an ³¹P NMR spectrum of synthesized compound 5.

FIG. 26 depicts an ¹H NMR spectrum of synthesized1-(2-(Pyrenesulfonyl)ethyl)-(β-dibenzyl)pyrophosphate (compound 6).

FIG. 27 depicts a ¹³C NMR spectrum of synthesized compound 6.

FIG. 28 depicts an ³¹P NMR spectrum of synthesized compound 6.

FIG. 29 depicts an ¹H NMR spectrum of synthesized compound 7.

FIG. 30 depicts a ¹³C NMR spectrum of synthesized compound 7.

FIG. 31 depicts an ³¹P NMR spectrum of synthesized compound 7.

FIG. 32 depicts a synthesis scheme for1-(2-(pyrenesulfonyl)ethyl)pyrophosphate (dTTP, compound 13a).

FIG. 33 depicts an ¹H NMR spectrum of synthesized3′-O-Benzoyl-2′-deoxythymidine-5′-dibenzylmonophosphate (compound 9a).

FIG. 34 depicts a ¹³C NMR spectrum of synthesized compound 9a.

FIG. 35 depicts an ³¹P NMR spectrum of synthesized compound 9a.

FIG. 36 depicts an ¹H NMR spectrum of synthesized3′-O-Benzoyl-2′-deoxythymidine-5′-monophosphate (compound 10a).

FIG. 37 depicts a ¹³C NMR spectrum of synthesized compound 10a.

FIG. 38 depicts an ³¹P NMR spectrum of synthesized compound 10a.

FIG. 39 depicts an ³¹P NMR spectrum of synthesized3′-O-Benzoyl-2′-deoxythymidine-5′-phosphor-2-methylimidazolide (compound11a).

FIG. 40 depicts an ¹H NMR spectrum of synthesized3′-O-Benzoyl-2′-deoxythymidine-5′-(γ-(2-(pyrenesulfonyl)ethyl))triphosphate(compound 12a).

FIG. 41 depicts an ³¹P NMR spectrum of synthesized compound 12a.

FIG. 42 depicts an 1H NMR spectrum of synthesized compound 13a.

FIG. 43 depicts a ³¹P NMR spectrum of synthesized compound 13a.

FIG. 44 depicts a synthesis scheme for 2′-deoxycytidine-5′-triphosphate(dCTP, compound βb).

FIG. 45 depicts an 1H NMR spectrum of synthesized 3′-O,N4-Dibenzoyl-2′-deoxycytidine-5′-dibenzylmonophosphate (compound 9b).

FIG. 46 depicts a ¹³C NMR spectrum of synthesized compound 9b.

FIG. 47 depicts an ³¹P NMR spectrum of synthesized compound 9b.

FIG. 48 depicts an 1H NMR spectrum of synthesized 3′-O,N4-Dibenzoyl-2′-deoxycytidine-5′-monophosphate (compound 10b).

FIG. 49 depicts a ¹³C NMR spectrum of synthesized compound 10b.

FIG. 50 depicts an ³¹P NMR spectrum of synthesized compound 10b.

FIG. 51 depicts an ³¹P NMR spectrum of synthesized 3′-O,N⁴-Dibenzoyl-2′-deoxycytidine-5′-phosphor-2-methylimidazolide (compound1 b).

FIG. 52 depicts an ¹H NMR spectrum of synthesized3′-O,N⁴-Dibenzoyl-2′-deoxycytidine-5′-(γ-(2-(pyrenesulfonyl)ethyl))triphosphate(compound 12b).

FIG. 53 depicts an ³¹P NMR spectrum of synthesized compound 12b.

FIG. 54 depicts an ¹H NMR spectrum of synthesized compound βb.

FIG. 55 depicts a ³¹P NMR spectrum of synthesized compound βb.

FIG. 56 depicts a synthesis scheme for 2′-deoxyadenosine-5′-triphosphate(dATP, compound βc).

FIG. 57 depicts an ¹H NMR spectrum of synthesized 3′-O, N⁶,N⁶-Tribenzoyl-2′-deoxyadenosine-5′-dibenzylmonophosphate (compound 9c).

FIG. 58 depicts a ¹³C NMR spectrum of synthesized compound 9c.

FIG. 59 depicts an ³¹P NMR spectrum of synthesized compound 9c.

FIG. 60 depicts an ¹H NMR spectrum of synthesized 3′-O, N⁶,N⁶-Tribenzoyl-2′-deoxyadenosine-5′-monophosphate (compound 10c).

FIG. 61 depicts a ¹³C NMR spectrum of synthesized compound 10c.

FIG. 62 depicts an ³¹P NMR spectrum of synthesized compound 10c.

FIG. 63 depicts an ³¹P NMR spectrum of synthesized 3′-O, N⁶,N⁶-Tribenzoyl-2′-deoxyadenosine-5′-phosphor-2-methylimidazolide(compound 11c).

FIG. 64 depicts an ¹H NMR spectrum of synthesized 3′-O, N⁶,N⁶-Tribenzoyl-2′-deoxyadenosine-5′-γ-(2-(pyrenesulfonyl)ethyl]-triphosphate(compound 12c).

FIG. 65 depicts an ³¹P NMR spectrum of synthesized compound 12c.

FIG. 66 depicts an ¹H NMR spectrum of synthesized compound βc.

FIG. 67 depicts a ³¹P NMR spectrum of synthesized compound βc.

FIG. 68 depicts a synthesis scheme for 2′-deoxyguanosine-5′-triphosphate(dGTP, compound βd).

FIG. 69 depicts an ¹H NMR spectrum of synthesized 3′-O,N2-Dibenzoyl-2′-deoxyguanosine-5′-dibenzylmonophosphate (compound 9d).

FIG. 70 depicts a ¹³C NMR spectrum of synthesized compound 9d.

FIG. 71 depicts an ³¹P NMR spectrum of synthesized compound 9d.

FIG. 72 depicts an ¹H NMR spectrum of synthesized 3′-O,N2-Dibenzoyl-2′-deoxyguanosine-5′-monophosphate (compound 10d).

FIG. 73 depicts a ¹³C NMR spectrum of synthesized compound 10d.

FIG. 74 depicts an ³¹P NMR spectrum of synthesized compound 10d.

FIG. 75 depicts an ³¹P NMR spectrum of synthesized 3′-O,N²-Dibenzoyl-2′-deoxyguanosine-5′-phosphor-2-methylimidazolide (compound11d).

FIG. 76 depicts an ¹H NMR spectrum of synthesized 3′-O,N²-Dibenzoyl-2′-deoxyadenosine-5′-(γ-(2-(pyrenesulfonyl)ethyl))-triphosphate(compound 12d).

FIG. 77 depicts an ³¹P NMR spectrum of synthesized compound 12d.

FIG. 78 depicts an ¹H NMR spectrum of synthesized compound βd.

FIG. 79 depicts a ³¹P NMR spectrum of synthesized compound βd.

FIG. 80 depicts a synthesis scheme for 2′-deoxythymidine-3′-triphosphate(3′-TTP, compound 19).

FIG. 81 depicts an ¹H NMR spectrum of synthesized5′-Benzoyl-2′-deoxythymidine-3′-dibenzylmonophosphate (compound 15).

FIG. 82 depicts a ¹³C NMR spectrum of synthesized compound 15.

FIG. 83 depicts an ³¹P NMR spectrum of synthesized compound 15.

FIG. 84 depicts an ¹H NMR spectrum of synthesized5′-Benzoyl-2′-deoxythymidine-3′-monophosphate (compound 16).

FIG. 85 depicts a ¹³C NMR spectrum of synthesized compound 16.

FIG. 86 depicts an ³¹P NMR spectrum of synthesized compound 16.

FIG. 87 depicts an ³¹P NMR spectrum of synthesized5′-Benzoyl-2′-deoxythymidine-3′-phosphor-2-methylimidazolide (compound17).

FIG. 88 depicts an ¹H NMR spectrum of synthesized5′-Benzoyl-2′-deoxythymidine-3′-γ-(2-(pyrenesulfonyl)ethyl)triphosphate(compound 18).

FIG. 89 depicts an ³¹P NMR spectrum of synthesized compound 18.

FIG. 90 depicts an ¹H NMR spectrum of synthesized compound 19.

FIG. 91 depicts a ³¹P NMR spectrum of synthesized compound 19.

FIG. 92 depicts a synthesis scheme for1-(α-L-threofuranosyl)thymidine-3′-triphosphate (tTTP, compound 25a).

FIG. 93 depicts an ¹H NMR spectrum of synthesized1-(2′-O-benzoyl-α-L-threofuranosyl)thymidine-3′-monophosphate (compound22a).

FIG. 94 depicts a ¹³C NMR spectrum of synthesized compound 22a.

FIG. 95 depicts an ³¹P NMR spectrum of synthesized compound 22a.

FIG. 96 depicts an ³¹P NMR spectrum of synthesized1-(2′-O-benzoyl-α-L-threofuranosyl)thymidine-3′-monophosphor-2-methylimidazolide(compound 23a).

FIG. 97 depicts an ¹H NMR spectrum of synthesized1-(2′-O-benzoyl-α-L-threofuranosyl)thymidine-3′-(γ-(2-(pyrenesulfonyl)ethyl))triphosphate (compound 24a).

FIG. 98 depicts an ³¹P NMR spectrum of synthesized compound 24a.

FIG. 99 depicts an ¹H NMR spectrum of synthesized compound 25a.

FIG. 100 depicts a ³¹P NMR spectrum of synthesized compound 25a.

FIG. 101 depicts a synthesis scheme for1-(α-L-threofuranosyl)cytidine-3′-triphosphate (tCTP, compound 25b).

FIG. 102 depicts an ¹H NMR spectrum of synthesizedN⁴-Benzoyl-1-(2′-O-benzoyl-α-L-threofuranosyl)cytidine-3′-monophosphate(compound 22b).

FIG. 103 depicts a ¹³C NMR spectrum of synthesized compound 22b.

FIG. 104 depicts an ³¹P NMR spectrum of synthesized compound 22b.

FIG. 105 depicts an ³¹P NMR spectrum of synthesizedN⁴-Benzoyl-1-(2′-O-benzoyl-α-L-threofuranosyl)cytidine-3′-monophosphor-2-methylimidazolide(compound 23b).

FIG. 106 depicts an ¹H NMR spectrum of synthesizedN⁴-Benzoyl-1-(2′-O-benzoyl-α-L-threofuranosyl)cytidine-3′-(γ-(2-(pyrenesulfonyl)ethyl)triphosphate (compound 24b).

FIG. 107 depicts an ³¹P NMR spectrum of synthesized compound 24b.

FIG. 108 depicts an ¹H NMR spectrum of synthesized compound 25b.

FIG. 109 depicts a ³¹P NMR spectrum of synthesized compound 25b.

FIG. 110 depicts a synthesis scheme for9-(α-L-threofuranosyl)adenosine-3′-triphosphate (tATP, compound 25c).

FIG. 111 depicts an ¹H NMR spectrum of synthesizedN⁶-Benzoyl-9-(2′-O-benzoyl-α-L-threofuranosyl)adenosine-3′-monophosphate(compound 22c).

FIG. 112 depicts a ¹³C NMR spectrum of synthesized compound 22c.

FIG. 113 depicts an ³¹P NMR spectrum of synthesized compound 22c.

FIG. 114 depicts an ³¹P NMR spectrum of synthesizedN⁶-Benzoyl-9-(2′-O-benzoyl-α-L-threofuranosyl)adenosine-3′-monophosphor-2-methylimidazolide(compound 23c).

FIG. 115 depicts an ¹H NMR spectrum of synthesizedN⁶-Benzoyl-9-(2′-O-benzoyl-α-L-threofuranosyl)adenosine-3′-γ-[2-(pyrenesulphonyl)ethyl]triphosphate(compound 24c).

FIG. 116 depicts an ³¹P NMR spectrum of synthesized compound 24c.

FIG. 117 depicts an ¹H NMR spectrum of synthesized compound 25c.

FIG. 118 depicts a ³¹P NMR spectrum of synthesized compound 25c.

FIG. 119 depicts a synthesis scheme for9-(α-L-threofuranosyl)guanosine-3′-triphosphate (tGTP, compound 25d).

FIG. 120 depicts an ¹H NMR spectrum of synthesizedN²-Acetyl-9-(2′-O-benzoyl-α-L-threofuranosyl)guanosine-3′-monophosphate(compound 22d).

FIG. 121 depicts a ¹³C NMR spectrum of synthesized compound 22d.

FIG. 122 depicts an ³¹P NMR spectrum of synthesized compound 22d.

FIG. 123 depicts an ³¹P NMR spectrum of synthesizedN²-Acetyl-9-(2′-O-benzoyl-α-L-threofuranosyl)guanosine-3′-monophosphor-2-methylimidazolide(compound 23d).

FIG. 124 depicts an ¹H NMR spectrum of synthesizedN²-Acetyl-9-(2′-O-benzoyl-α-L-threofuranosyl)guanosine-3′-(γ-(2-(pyrenesulfonyl)ethyl))triphosphate (compound 24d).

FIG. 125 depicts an ³¹P NMR spectrum of synthesized compound 24d.

FIG. 126 depicts an ¹H NMR spectrum of synthesized compound 25d.

FIG. 127 depicts a ³¹P NMR spectrum of synthesized compound 25d.

FIG. 128 depicts a synthesis scheme forL-2′-deoxythymidine-5′-triphosphate (L-dTTP, compound 31).

FIG. 129 depicts an ¹H NMR spectrum of synthesized3′-Benzoyl-2′-deoxy-L-thymidine-5′-dibenzylmonophosphate (compound 27).

FIG. 130 depicts a ¹³C NMR spectrum of synthesized compound 27.

FIG. 131 depicts an ³¹P NMR spectrum of synthesized compound 27.

FIG. 132 depicts an ¹H NMR spectrum of synthesized3′-Benzoyl-2′-deoxy-L-thymidine-5′-monophosphate (compound 28).

FIG. 133 depicts a ¹³C NMR spectrum of synthesized compound 28.

FIG. 134 depicts an ³¹P NMR spectrum of synthesized compound 28.

FIG. 135 depicts an ³¹P NMR spectrum of synthesized3′-Benzoyl-2′-deoxy-L-thymidine-5′-phosphor-2-methylimidazolide(compound 29).

FIG. 136 depicts an ¹H NMR spectrum of synthesized3′-Benzoyl-2′-deoxy-L-thymidine-5′-(γ-(2-(pyrenesulfonyl)ethyl))triphosphate(compound 30).

FIG. 137 depicts an ³¹P NMR spectrum of synthesized compound 30.

FIG. 138 depicts an ¹H NMR spectrum of synthesized compound 31.

FIG. 139 depicts a ³¹P NMR spectrum of synthesized compound 31.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery that anorganic molecule comprising at least one phosphate moiety can be used asa phosphorylation reagent. In one embodiment, the organic moleculecomprising at least one phosphate moiety can be used as aphosphorylation reagent to synthesize a phosphorylated molecule througha reaction of the organic molecule comprising at least one phosphatemoiety with an activated precursor molecule. The compositions andmethods disclosed herein can be used to synthesize phosphorylated sugarmolecules, phosphorylated proteins or peptides, phosphorylated lipids,or phosphorylated nucleic acid molecules. Thus, in various aspects theinvention provides compositions and methods for synthesizing natural andmodified phosphorylated molecules.

The compositions and methods disclosed herein can be used to synthesizephosphorylated nucleosides. In one embodiment, the organic phosphatemolecule of the invention can be an organic pyrophosphate molecule foruse in the synthesis of nucleoside triphosphates from an activatednucleoside monophosphate molecule. In one embodiment, the organicphosphate molecule of the invention can be an organic monophosphatemolecule for use in the synthesis of nucleoside diphosphates from anactivated nucleoside monophosphate molecule. In one embodiment, theorganic phosphate molecule of the invention can be an organictriphosphate molecule for use in the synthesis of nucleosidetetraphosphates from an activated nucleoside monophosphate molecule. Inone embodiment, the organic phosphate molecule of the invention can bean organic tetraphosphate molecule for use in the synthesis ofnucleoside pentaphosphates from an activated nucleoside monophosphatemolecule. In one embodiment, the organic phosphate molecule of theinvention can be an organic pentaphosphate molecule for use in thesynthesis of nucleoside hexaphosphates from an activated nucleosidemonophosphate molecule.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassnon-limiting variations of ±40% or ±20% or ±10%, ±5%, ±1%, or ±0.1% fromthe specified value, as such variations are appropriate.

“Acid catalyst,” as used herein, refers to any acidic compoundsincluding the so-called Lewis acids, which catalyze the reaction betweenan activated nucleoside monophosphate and a pyrophosphate containingcompound. Examples of acids used for this purpose include ZnCl₂, H₂SO₄,HCl, H₃PO₄ or BF₃, or sulfonic acids or their salts. Examples ofsulfonic acids comprise ortho-, meta- and para-toluenesulfonic acids,alkylbenzenesulfonic acids, secondary alkyl-sulfonic acids, sulfonicresins, alkylsulfates, alkylbenzenesulfonates, alkyl-sulfonates andsulfosuccinic acid.

As used herein, the term “purified” or “to purify” refers to the removalof components (e.g., contaminants) from a sample. For example, nucleicacids are purified by removal of contaminating cellular proteins orother undesired nucleic acid species. The removal of contaminantsresults in an increase in the percentage of a desired compound in thesample.

A “natural” nucleoside is one that occurs in nature. For the purposes ofthis invention the following nucleosides are defined as the naturalnucleosides: adenosine, cytidine, guanosine, uridine, 2′-deoxyadenosine,2′-deoxycytidine, 2′-deoxyguanosine, thymidine, and inosine.

The term base, unless otherwise specified, refers to the base moiety ofa nucleoside or nucleotide (a nucleobases). The base moiety is theheterocycle portion of a nucleoside or nucleotide. The base moiety maybe a pyrimidine derivative or analog, a purine derivative or analog, orother heterocycle. The nucleoside base may contain two or more nitrogenatoms and may contain one or more peripheral substitutents. Thenucleoside base is attached to the sugar moiety of the nucleotide mimicin such ways that both β-D- and β-L-nucleoside and nucleotide can beproduced.

The term sugar refers to the ribofuranose of deoxyribofuranose portionof a nucleoside or nucleotide. The sugar moiety may contain one or moresubstitutents at the C1-, C2-, C3-, C4-, and C5-position of theribofuranose. Substituents may direct to either the α- or β-face of theribofuranose. The nucleoside base that can be considered as asubstitutent at the C-1 position of the ribofuranose directs to theβ-face of the sugar. The β-face is the side of a ribofuranose on which apurine or pyrimidine base of natural β-D-nucleosides is present. Theα-face is the side of the sugar opposite to the β-face. The sugar moietyof the present invention is not limited to a ribofuranose and itsderivatives, instead, it may be a carbohydrate, a carbohydrate analog, acarbocyclic ring, or other ribofuranose analog.

The term sugar-modified nucleoside refers to a nucleoside containing amodified sugar moiety.

The term base-modified nucleoside refers to a nucleoside containing amodified base moiety, relative to a base moiety found in a naturalnucleoside.

As used herein, the term “nucleic acid” refers to bothnaturally-occurring molecules such as DNA and RNA, but also variousderivatives and analogs. Generally, the probes, hairpin linkers, andtarget polynucleotides of the present teachings are nucleic acids, andtypically comprise DNA. Additional derivatives and analogs can beemployed as will be appreciated by one having ordinary skill in the art.

The term “nucleotide base”, as used herein, refers to a substituted orunsubstituted aromatic ring or rings. In certain embodiments, thearomatic ring or rings contain at least one nitrogen atom. In certainembodiments, the nucleotide base is capable of forming Watson-Crickand/or Hoogsteen hydrogen bonds with an appropriately complementarynucleotide base. Exemplary nucleotide bases and analogs thereof include,but are not limited to, naturally occurring nucleotide bases adenine,guanine, cytosine, 6 methyl-cytosine, uracil, thymine, and analogs ofthe naturally occurring nucleotide bases, e.g., 7-deazaadenine,7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine, N6 delta2-isopentenyladenine (6iA), N6-delta 2-isopentenyl-2-methylthioadenine(2 ms6iA), N2-dimethylguanine (dmG), 7methylguanine (7mG), inosine,nebularine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine,hypoxanthine, pseudouridine, pseudocytosine, pseudoisocytosine,5-propynylcytosine, isocytosine, isoguanine, 7-deazaguanine,2-thiopyrimidine, 6-thioguanine, 4-thiothymine, 4-thiouracil,06-methylguanine, N6-methyladenine, 04-methylthymine,5,6-dihydrothymine, 5,6-dihydrouracil, pyrazolo[3,4-D]pyrimidines (see,e.g., U.S. Pat. Nos. 6,143,877 and 6,127,121 and PCT publishedapplication WO 01/38584), ethenoadenine, indoles such as nitroindole and4-methylindole, and pyrroles such as nitropyrrole. Certain exemplarynucleotide bases can be found, e.g., in Fasman, 1989, Practical Handbookof Biochemistry and Molecular Biology, pp. 385-394, CRC Press, BocaRaton, Fla., and the references cited therein.

The term “nucleotide”, as used herein, refers to a compound comprising anucleotide base linked to the C-1′ carbon of a sugar, such as ribose,arabinose, xylose, and pyranose, and sugar analogs thereof. The termnucleotide also encompasses nucleotide analogs. The sugar may besubstituted or unsubstituted. Substituted ribose sugars include, but arenot limited to, those riboses in which one or more of the carbon atoms,for example the 2′-carbon atom, is substituted with one or more of thesame or different Cl, F, —R, —OR, —NR₂ or halogen groups, where each Ris independently H, C1-C6 alkyl or C5-C14 aryl. Exemplary ribosesinclude, but are not limited to, 2′-(C1-C6)alkoxyribose,2′-(C5-C14)aryloxyribose, 2′,3′-didehydroribose, 2′-deoxy-3′-haloribose,2′-deoxy-3′-fluororibose, 2′-deoxy-3′-chlororibose,2′-deoxy-3′-aminoribose, 2′-deoxy-3′-(C1-C6)alkylribose,2′-deoxy-3′-(C1-C6)alkoxyribose and 2′-deoxy-3′-(C5-C14)aryloxyribose,ribose, 2′-deoxyribose, 2′,3′-dideoxyribose, 2′-haloribose,2′-fluororibose, 2′-chlororibose, and 2′-alkylribose, e.g., 2′-O-methyl,4′-anomeric nucleotides, 1′-anomeric nucleotides, 2′-4′- and3′-4′-linked and other “locked” or “LNA”, bicyclic sugar modifications(see, e.g., PCT published application nos. WO 98/22489, WO 98/39352; andWO 99/14226). The term “nucleic acid” typically refers to largepolynucleotides.

The term “nucleotide analogs” as used herein refers to modified ornon-naturally occurring nucleotides including, but not limited to,analogs that have altered stacking interactions such as 7-deaza purines(i.e., 7-deaza-dATP and 7-deaza-dGTP); base analogs with alternativehydrogen bonding configurations (e.g., such as Iso-C and Iso-G and othernon-standard base pairs described in U.S. Pat. No. 6,001,983 to S.Benner and herein incorporated by reference); non-hydrogen bondinganalogs (e.g., non-polar, aromatic nucleoside analogs such as2,4-difluorotoluene, described by B. A. Schweitzer and E. T. Kool, J.Org. Chem., 1994, 59, 7238-7242; B. A. Schweitzer and E. T. Kool, J. Am.Chem. Soc., 1995, 117, 1863-1872); “universal” bases such as5-nitroindole and 3-nitropyrrole; and universal purines and pyrimidines(such as “K” and “P” nucleotides, respectively; P. Kong, et al., NucleicAcids Res., 1989, 17, 10373-10383, P. Kong et al., Nucleic Acids Res.,1992, 20, 5149-5152). Nucleotide analogs include nucleotides havingmodification on the sugar moiety, such as dideoxy nucleotides and2′-O-methyl nucleotides. Nucleoside analogue examples wherein thenatural sugar moiety is modified include but are not limited to hexitolnucleic acid (HNA), cyclohexene nucleic acids (CeNA), locked nucleicacids (LNA), altritol nucleic acids (ANA) and peptide nucleic acids(PNA). Nucleotide analogs include modified forms ofdeoxyribo-nucleotides as well as ribonucleotides.

The term “oligonucleotide” typically refers to short polynucleotides,generally, no greater than about 50 nucleotides. It will be understoodthat when a nucleotide sequence is represented by a DNA sequence (i.e.,A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) inwhich “U” replaces “T.” The term “polynucleotide” as used herein isdefined as a chain of nucleotides. Furthermore, nucleic acids arepolymers of nucleotides. Thus, nucleic acids and polynucleotides as usedherein are interchangeable. One skilled in the art has the generalknowledge that nucleic acids are polynucleotides, which can behydrolyzed into the monomeric “nucleotides.” The monomeric nucleotidescan be hydrolyzed into nucleosides. As used herein polynucleotidesinclude, but are not limited to, all nucleic acid sequences which areobtained by any means available in the art, including, withoutlimitation, recombinant means, i.e., the cloning of nucleic acidsequences from a recombinant library or a cell genome, using ordinarycloning and amplification technology, and the like, and by syntheticmeans. An “oligonucleotide” as used herein refers to a shortpolynucleotide, typically less than 100 bases in length.

As used herein, the term “pharmaceutical composition” refers to amixture of at least one compound useful within the invention with apharmaceutically acceptable carrier. The pharmaceutical compositionfacilitates administration of the compound to a patient or subject.Multiple techniques of administering a compound exist in the artincluding, but not limited to, intravenous, oral, aerosol, parenteral,ophthalmic, pulmonary and topical administration.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology, for the purpose of diminishing oreliminating those signs.

As used herein, the term “treatment” or “treating” is defined as theapplication or administration of a therapeutic agent, i.e., a compoundof the invention (alone or in combination with another pharmaceuticalagent), to a patient, or application or administration of a therapeuticagent to an isolated tissue or cell line from a patient (e.g., fordiagnosis or ex vivo applications), who has a condition contemplatedherein, a sign or symptom of a condition contemplated herein or thepotential to develop a condition contemplated herein, with the purposeto cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve oraffect a condition contemplated herein, the symptoms of a conditioncontemplated herein or the potential to develop a condition contemplatedherein. Such treatments may be specifically tailored or modified, basedon knowledge obtained from the field of pharmacogenomics.

As used herein, the terms “effective amount,” “pharmaceuticallyeffective amount” and “therapeutically effective amount” refer to anontoxic but sufficient amount of an agent to provide the desiredbiological result. That result may be reduction and/or alleviation of asign, a symptom, or a cause of a disease or disorder, or any otherdesired alteration of a biological system. An appropriate therapeuticamount in any individual case may be determined by one of ordinary skillin the art using routine experimentation.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound, and is relativelynon-toxic, i.e., the material may be administered to an individualwithout causing an undesirable biological effect or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, the language “pharmaceutically acceptable salt” refersto a salt of the administered compound prepared from pharmaceuticallyacceptable non-toxic acids, including inorganic acids, organic acids,solvates, hydrates, or clathrates thereof. Examples of such inorganicacids are hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric,phosphoric, acetic, hexafluorophosphoric, citric, gluconic, benzoic,propionic, butyric, sulfosalicylic, maleic, lauric, malic, fumaric,succinic, tartaric, amsonic, pamoic, p-tolunenesulfonic, and mesylic.Appropriate organic acids may be selected, for example, from aliphatic,aromatic, carboxylic and sulfonic classes of organic acids, examples ofwhich are formic, acetic, propionic, succinic, camphorsulfonic, citric,fumaric, gluconic, isethionic, lactic, malic, mucic, tartaric,para-toluenesulfonic, glycolic, glucuronic, maleic, furoic, glutamic,benzoic, anthranilic, salicylic, phenylacetic, mandelic, embonic(pamoic), methanesulfonic, ethanesulfonic, pantothenic, benzenesulfonic(besylate), stearic, sulfanilic, alginic, galacturonic, and the like.Furthermore, pharmaceutically acceptable salts include, by way ofnon-limiting example, alkaline earth metal salts (e.g., calcium ormagnesium), alkali metal salts (e.g., sodium-dependent or potassium),and ammonium salts.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound useful within theinvention within or to the patient such that it may perform its intendedfunction. Typically, such constructs are carried or transported from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation, including the compound usefulwithin the invention, and not injurious to the patient. Some examples ofmaterials that may serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; surface active agents; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffersolutions; and other non-toxic compatible substances employed inpharmaceutical formulations. As used herein, “pharmaceuticallyacceptable carrier” also includes any and all coatings, antibacterialand antifungal agents, and absorption delaying agents, and the like thatare compatible with the activity of the compound useful within theinvention, and are physiologically acceptable to the patient.Supplementary active compounds may also be incorporated into thecompositions. The “pharmaceutically acceptable carrier” may furtherinclude a pharmaceutically acceptable salt of the compound useful withinthe invention. Other additional ingredients that may be included in thepharmaceutical compositions used in the practice of the invention areknown in the art and described, for example in Remington'sPharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton,Pa.), which is incorporated herein by reference.

As used herein, the term “alkyl,” by itself or as part of anothersubstituent means, unless otherwise stated, a straight or branched chainhydrocarbon having the number of carbon atoms designated (i.e. C1-6means one to six carbon atoms) and including straight, branched chain,or cyclic substituent groups. Examples include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, andcyclopropylmethyl.

As used herein, the term “substituted alkyl” means alkyl as definedabove, substituted by one, two or three substituents selected from thegroup consisting of halogen, —OH, alkoxy, —NH₂, amino, azido, —N(CH₃)₂,—C(═O)OH, trifluoromethyl, —C≡N, —C(═O)O(C₁-C₄)alkyl, —C(═O)NH₂,—SO₂NH₂, —C(═NH)NH₂, and —NO₂. Examples of substituted alkyls include,but are not limited to, 2,2-difluoropropyl, 2-carboxycyclopentyl and3-chloropropyl.

As used herein, the term “heteroalkyl” by itself or in combination withanother term means, unless otherwise stated, a stable straight orbranched chain alkyl group consisting of the stated number of carbonatoms and one or two heteroatoms selected from the group consisting ofO, N, and S, and wherein the nitrogen and sulfur atoms may be optionallyoxidized and the nitrogen heteroatom may be optionally quaternized. Theheteroatom(s) may be placed at any position of the heteroalkyl group,including between the rest of the heteroalkyl group and the fragment towhich it is attached, as well as attached to the most distal carbon atomin the heteroalkyl group. Examples include: —O—CH₂—CH₂—CH₃,—CH₂—CH₂—CH₂—OH, —CH₂—CH₂—NH—CH₃, —CH₂—S—CH₂—CH₃, and —CH₂CH₂—S(═O)—CH₃.Up to two heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃, or —CH₂—CH₂—S—S—CH₃ As used herein, the term “alkoxy”employed alone or in combination with other terms means, unlessotherwise stated, an alkyl group having the designated number of carbonatoms, as defined above, connected to the rest of the molecule via anoxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy(isopropoxy) and the higher homologs and isomers.

As used herein, the term “halo” or “halogen” alone or as part of anothersubstituent means, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom.

As used herein, the term “cycloalkyl” refers to a mono cyclic orpolycyclic non-aromatic radical, wherein each of the atoms forming thering (i.e. skeletal atoms) is a carbon atom. In one embodiment, thecycloalkyl group is saturated or partially unsaturated. In anotherembodiment, the cycloalkyl group is fused with an aromatic ring.Cycloalkyl groups include groups having from 3 to 10 ring atoms.Illustrative examples of cycloalkyl groups include, but are not limitedto, the following moieties:

Monocyclic cycloalkyls include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.Dicyclic cycloalkyls include, but are not limited to,tetrahydronaphthyl, indanyl, and tetrahydropentalene. Polycycliccycloalkyls include adamantine and norbornane. The term cycloalkylincludes “unsaturated nonaromatic carbocyclyl” or “nonaromaticunsaturated carbocyclyl” groups, both of which refer to a nonaromaticcarbocycle as defined herein, which contains at least one carbon doublebond or one carbon triple bond.

As used herein, the term “heterocycloalkyl” or “heterocyclyl” refers toa heteroalicyclic group containing one to four ring heteroatoms eachselected from O, S and N. In one embodiment, each heterocycloalkyl grouphas from 4 to 10 atoms in its ring system, with the proviso that thering of said group does not contain two adjacent O or S atoms. Inanother embodiment, the heterocycloalkyl group is fused with an aromaticring. In one embodiment, the nitrogen and sulfur heteroatoms may beoptionally oxidized, and the nitrogen atom may be optionallyquaternized. The heterocyclic system may be attached, unless otherwisestated, at any heteroatom or carbon atom that affords a stablestructure. A heterocycle may be aromatic or non-aromatic in nature. Inone embodiment, the heterocycle is a heteroaryl.

An example of a 3-membered heterocycloalkyl group includes, and is notlimited to, aziridine. Examples of 4-membered heterocycloalkyl groupsinclude, and are not limited to, azetidine and a beta lactam. Examplesof 5-membered heterocycloalkyl groups include, and are not limited to,pyrrolidine, oxazolidine and thiazolidinedione. Examples of 6-memberedheterocycloalkyl groups include, and are not limited to, piperidine,morpholine and piperazine. Other non-limiting examples ofheterocycloalkyl groups are:

Examples of non-aromatic heterocycles include monocyclic groups such asaziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine,pyrroline, pyrazolidine, imidazoline, dioxolane, sulfolane,2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane,piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine,morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran,1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane,4,7-dihydro-1,3-dioxepin, and hexamethyleneoxide.

As used herein, the term “aromatic” refers to a carbocycle orheterocycle with one or more polyunsaturated rings and having aromaticcharacter, i.e. having (4n+2) delocalized π (pi) electrons, where n isan integer.

As used herein, the term “aryl,” employed alone or in combination withother terms, means, unless otherwise stated, a carbocyclic aromaticsystem containing one or more rings (typically one, two or three rings),wherein such rings may be attached together in a pendent manner, such asa biphenyl, or may be fused, such as naphthalene. Examples of arylgroups include phenyl, anthracyl, and naphthyl.

As used herein, the term “aryl-(C₁-C₃)alkyl” means a functional groupwherein a one- to three-carbon alkylene chain is attached to an arylgroup, e.g., —CH₂CH₂-phenyl. In one embodiment, aryl-(C₁-C₃)alkyl isaryl-CH₂— or aryl-CH(CH₃)—. The term “substituted aryl-(C₁-C₃)alkyl”means an aryl-(C₁-C₃)alkyl functional group in which the aryl group issubstituted. Similarly, the term “heteroaryl-(C₁-C₃)alkyl” means afunctional group wherein a one to three carbon alkylene chain isattached to a heteroaryl group, e.g., —CH₂CH₂-pyridyl. The term“substituted heteroaryl-(C₁-C₃)alkyl” means a heteroaryl-(C₁-C₃)alkylfunctional group in which the heteroaryl group is substituted.

As used herein, the term “heteroaryl” or “heteroaromatic” refers to aheterocycle having aromatic character. A polycyclic heteroaryl mayinclude one or more rings that are partially saturated. Examples includethe following moieties:

Examples of heteroaryl groups also include pyridyl, pyrazinyl,pyrimidinyl (particularly 2- and 4-pyrimidinyl), pyridazinyl, thienyl,furyl, pyrrolyl (particularly 2-pyrrolyl), imidazolyl, thiazolyl,oxazolyl, pyrazolyl (particularly 3- and 5-pyrazolyl), isothiazolyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl,1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and1,3,4-oxadiazolyl.

Examples of polycyclic heterocycles and heteroaryls include indolyl(particularly 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl,tetrahydroquinolyl, isoquinolyl (particularly 1- and 5-isoquinolyl),1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2-and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl,1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl,benzofuryl (particularly 3-, 4-, 5-, 6- and 7-benzofuryl),2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (particularly3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl(particularly 2-benzothiazolyl and 5-benzothiazolyl), purinyl,benzimidazolyl (particularly 2-benzimidazolyl), benzotriazolyl,thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, andquinolizidinyl.

As used herein, the term “substituted” means that an atom or group ofatoms has replaced hydrogen as the substituent attached to anothergroup. The term “substituted” further refers to any level ofsubstitution, namely mono-, di-, tri-, tetra-, or penta-substitution,where such substitution is permitted. The substituents are independentlyselected, and substitution may be at any chemically accessible position.In one embodiment, the substituents vary in number between one and four.In another embodiment, the substituents vary in number between one andthree. In yet another embodiment, the substituents vary in numberbetween one and two.

As used herein, the term “optionally substituted” means that thereferenced group may be substituted or unsubstituted. In one embodiment,the referenced group is optionally substituted with zero substituents,i.e., the referenced group is unsubstituted. In another embodiment, thereferenced group is optionally substituted with one or more additionalgroup(s) individually and independently selected from groups describedherein.

In one embodiment, the substituents are independently selected from thegroup consisting of oxo, halogen, —CN, —NH₂, —OH, —NH(CH₃), —N(CH₃)₂,alkyl (including straight chain, branched and/or unsaturated alkyl),substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, fluoro alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted alkoxy, fluoroalkoxy,—S-alkyl, S(═O)₂alkyl, —C(═O)NH[substituted or unsubstituted alkyl, orsubstituted or unsubstituted phenyl], —C(═O)N[H or alkyl]₂,—OC(═O)N[substituted or unsubstituted alkyl]₂, —NHC(═O)NH[substituted orunsubstituted alkyl, or substituted or unsubstituted phenyl],—NHC(═O)alkyl, —N[substituted or unsubstituted alkyl]C(═O)[substitutedor unsubstituted alkyl], —NHC(═O)[substituted or unsubstituted alkyl],—C(OH)[substituted or unsubstituted alkyl]₂, and —C(NH₂)[substituted orunsubstituted alkyl]₂. In another embodiment, by way of example, anoptional substituent is selected from oxo, fluorine, chlorine, bromine,iodine, —CN, —NH₂, —OH, —NH(CH₃), —N(CH₃)₂, —CH₃, —CH₂CH₃, —CH(CH₃)₂,—CF₃, —CH₂CF₃, —OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, —OCF₃, —OCH₂CF₃,—S(═O)₂—CH₃, —C(═O)NH₂, —C(═O)—NHCH₃, —NHC(═O)NHCH₃, —C(═O)CH₃, —ON(O)₂,and —C(═O)OH. In yet one embodiment, the substituents are independentlyselected from the group consisting of C₁₋₆ alkyl, —OH, C₁₋₆ alkoxy,halo, amino, acetamido, oxo and nitro. In yet another embodiment, thesubstituents are independently selected from the group consisting ofC₁₋₆ alkyl, C₁₋₆ alkoxy, halo, acetamido, and nitro. As used herein,where a substituent is an alkyl or alkoxy group, the carbon chain may bebranched, straight or cyclic.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

In one embodiment, the invention provides organic molecules comprisingat least one phosphate moiety, and methods of use for the synthesis ofphosphorylated molecules. The phosphorylated molecule generated usingthe compositions and methods of the invention may be a phosphorylatedprotein, a phosphorylated sugar, a phosphorylated lipid, or aphosphorylated nucleoside molecule. Phosphorylated nucleosides that canbe synthesized using the methods of the invention include, but are notlimited to, natural or modified nucleoside hexaphosphates (e.g., 5′nucleoside hexaphosphate (Np₆) and 2′-deoxynucleoside-5′-hexaphosphates(dNp₆)), nucleoside hexaphosphate analogs, natural or modifiednucleoside pentaphosphates (e.g., pppGpp, dinucleoside pentaphosphate(e.g., diadenosine pentaphosphate), 5′ nucleoside pentaphostphate (Np₅)and 2′-deoxynucleoside-5′-pentaphosphates (dNp₅)), nucleosidepentaphosphate analogs, natural or modified nucleoside tetraphosphates(e.g., ppGpp, dinucleoside tetraphosphate (e.g., diadenosinetetraphosphate), 5′ nucleoside tetraphostphate (Np₄) and2′-deoxynucleoside-5′-tetraphosphates (dNp₄)), nucleoside tetraphosphateanalogs, natural or modified nucleoside triphosphates (e.g., 5′nucleoside triphostphate (NTPs), 5′ nucleoside triphostphate (NTPs) and2′-deoxynucleoside-5′-triphosphates (dNTPs)), nucleoside triphosphateanalogs, natural or modified nucleoside diphosphates (e.g., 5′nucleoside diphostphate (NDPs) and 2′-deoxynucleoside-5′-diphosphates(dNDPs)), nucleoside diphosphate analogs, natural or modified nucleosidemonophosphates (e.g., 5′ nucleoside monophostphate (NMPs) and2′-deoxynucleoside-5′-monophosphates (dNMPs)), and nucleosidemonophosphate analogs.

Compounds of the Invention The compounds of the present invention may besynthesized using techniques well-known in the art of organic synthesis.The starting materials and intermediates required for the synthesis maybe obtained from commercial sources or synthesized according to methodsknown to those skilled in the art.

In one embodiment, the invention relates to a compound represented byformula (1):

wherein, m is an integer from 0 to 5;

n is an integer from 0 to 5;

X is O or CH₂;

R₁₁ is an aryl, a heteroaryl, an alkyl, a cycloalkyl, an alkenyl, analkynyl, an alkyl-aryl, an aryl-alkyl, or a silyl group;

R₁₂ is hydrogen, null, a substituted tetahydrofuranyl group, or analkyl-substituted tetahydrofuranyl group; and

each occurrence of R₁₃ is independently hydrogen or null.

In one embodiment, R₁₁ is substituted with a group selected from thegroup consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heteroaryl, halogen, —CN, —OR₁₄, and —N(R₁₄)₂, wherein each occurrenceof R₁₄ is independently selected from the group consisting hydrogen,alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, and halogen.

In one embodiment, R₁₁ is selected from the group consisting of

In one embodiment, R₁₂ is hydrogen. In one embodiment, R₁₂ is null. Inone embodiment, when R₁₂ is null there is an anion. In one embodiment,R₁₂ is a methyl-tetrahydrofurnayl group wherein the tetrahydrofurnaylgroup is substituted. In one embodiment, R₁₂ is tetrahydrofurnayl groupwherein the tetrahydrofurnayl group is substituted. In one embodiment,the tetrahydrofurnayl group is substituted with a oxy-phenyl. In oneembodiment, the tetrahydrofurnayl group is substituted with anitrogenous base.

In one embodiment, each occurrence of R₁₃ is hydrogen. In one embodimentoccurrence of R₁₃ is null. In one embodiment, when R₁₃ is null there isan anion.

In one embodiment, m is 0.

In one embodiment m is 1.

In one embodiment, m is 2.

In one embodiment, m is 3.

In one embodiment, n is 0.

In one embodiment, n is 1.

In one embodiment, n is 2.

In one embodiment, n is 3.

In one embodiment, n is 4.

In one embodiment, n is 5.

In one embodiment, X is O.

In one embodiment, X is CH₂.

In one embodiment, the compound of formula (1) is a compound of formula(1a):

wherein R_(1a) is selected from the group consisting of an aryl, aheteroaryl, an alkyl, a cycloalkyl, an alkenyl, an alkynyl, analkyl-aryl, and an aryl-alkyl, wherein R_(1a) is optionally substituted.

In one embodiment, R_(1a) is substituted with a group selected from thegroup consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heteroaryl, halogen, —CN, —OR_(11a), and —N(R_(11a))₂, wherein eachoccurrence of R_(11a) is independently selected from the groupconsisting hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heteroaryl, and halogen.

In one embodiment, R_(1a) is selected from the group consisting of

In one embodiment, the compound of formula (1a) is selected from thegroup consisting of

In one embodiment, the compound of formula (1) is a compound of formula(1b):

wherein R_(1b) is a hydroxy protecting group.

In one embodiment, R_(1b) is selected from the group consisting of anaryl, a heteroaryl, an alkyl, a cycloalkyl, an alkenyl, an alkynyl, analkyl-aryl, and an aryl-alkyl, wherein R_(1b) is optionally substituted.

In one embodiment, R_(1b) is substituted with a group selected from thegroup consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heteroaryl, halogen, —CN, —OR_(11b), and —N(R_(11b))₂, wherein eachoccurrence of R_(11b) is independently selected from the groupconsisting hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heteroaryl, and halogen.

In one embodiment, R_(1b) is selected from the group consisting of

In one embodiment, the compound of formula (1b) is selected from thegroup consisting of

In one embodiment, the compound of formula (1) is a compound of formula(1c):

wherein:

m is an integer from 0 to 5;

n is an integer from 0 to 5;

X is O or CH₂;

R_(11c) is an aryl, a heteroaryl, an alkyl, a cycloalkyl, an alkenyl, analkynyl, an alkyl-aryl, an aryl-alkyl, or a silyl group;

each occurrence of Rhd 13 c is independently hydrogen or null;

R_(15c) is hydrogen, aryl, or heteroaryl; and

R_(16c) is a nitrogenous base, wherein the nitrogenous base is a naturalnitrogenous base or artificial nitrogenous base.

In one embodiment, R_(11c) is

In one embodiment, R_(15c) is phenyl.

In one embodiment the compound of formula (1c) is:

In one embodiment, the compound of formula (1) is a compound of formula(1d):

wherein:

m is an integer from 0 to 5;

n is an integer from 0 to 5;

X is O or CH₂;

R_(11d) is an aryl, a heteroaryl, an alkyl, a cycloalkyl, an alkenyl, analkynyl, an alkyl-aryl, an aryl-alkyl, or a silyl group;

each occurrence of R_(13d) is independently hydrogen or null;

R_(15d) is hydrogen, aryl, or heteroaryl; and

R_(16d) is a nitrogenous base, wherein the nitrogenous base is a naturalnitrogenous base or artificial nitrogenous base.

In one embodiment, R_(11d) is

In one embodiment, R_(15d) is phenyl.

In one embodiment, the compound of formula (1d) is:

In one embodiment, the compound of formula (1) is a compound of formula(β):

wherein:

m is an integer from 0 to 5;

n is an integer from 0 to 5;

X is O or CH₂;

R_(11e) is an aryl, a heteroaryl, an alkyl, a cycloalkyl, an alkenyl, analkynyl, an alkyl-aryl, an aryl-alkyl, or a silyl group;

each occurrence of R_(13e) is independently hydrogen or null;

R_(15e) is hydrogen, aryl, acetyl, or heteroaryl; and

R_(16e) is a nitrogenous base, wherein the nitrogenous base is a naturalnitrogenous base or artificial nitrogenous base.

In one embodiment, R_(11e) is

In one embodiment, R_(15e) is phenyl.

In one embodiment, the compound of formula (1e) is:

In one embodiment, the invention relates to a compound represented byformula (2):

wherein n is an integer from 1 to 10, and

R₂ is a support.

In one embodiment, the invention relates to a compound represented byformula (3):

wherein p is an integer from 0 to 10, and

R₃ is a support.

In one embodiment, the invention relates to a compound represented byformula (4):

wherein q is an integer from 0 to 10, and

r is an integer from 0 to 10.

In one embodiment q is 1. In one embodiment r is 1.

In one embodiment, the compound of formula (4) is

Supports

The attachment of useful materials such as catalysts, reagents,chelating or complexing agents, and proteins to insoluble supports iswell-known. With the attending advantages of ease of removal andrecovery from the system, e.g., by simple filtration, regeneration (ifnecessary), and recycling coupled with the increased utilization ofcontinuous flow systems in both general chemical processing anddiagnostic monitoring procedures, supported materials and methods ofgenerating polymer supported reaction reagents are well known in theart.

In one embodiment, the invention relates to a compound represented byformula (2) or formula (3) attached to an inorganic polymer support.Inorganic polymer supports include, but are not limited to, silica geland alumina.

In one embodiment, the invention relates to a compound represented byformula (2) or formula (3) attached to an organic polymer support.Organic polymer supports include, but are not limited to, polystyrene.

In one embodiment, the invention relates to a compound represented byformula (2) or formula (3) attached to solid support (e.g., controlledpore glass).

Nucleoside Triphosphates and Nucleic Acids

In one embodiment, the compounds and methods of the invention are usedto synthesize a naturally occurring nucleoside triphosphate. Naturallyoccurring nucleoside triphosphates include, but are not limited to,adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidinetriphosphate (CTP), 5-methyluridine triphosphate (m⁵UTP), and uridinetriphosphate (UTP). The terms ATP, GTP, CTP, and UTP refer to thosenucleoside triphosphates that contain ribose. The nucleosidetriphosphates containing deoxyribose are called dNTPs, and includedeoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP),deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP)and deoxyuridine triphosphate (dUTP).

In one embodiment, the compounds and methods of the invention are usedto synthesize isomers or analogs of nucleoside triphosphates. Exemplarynucleoside triphosphate analogs or isomers include, but are not limitedto, 2′-deoxythymidine-3′-triphosphate (3′-TTP),1-(α-L-threofuranosyl)thymidine-3′-triphosphate (tTTP),1-(α-L-threofuranosyl)cytidine-3′-triphosphate (tCTP),9-(α-L-threofuranosyl)adenosine-3′-triphosphate (tATP),9-(α-L-threofuranosyl)guanosine-3′-triphosphate (tGTP), andL-2′-deoxythymidine-5′-triphosphate (L-dTTP).

In some embodiments, the nucleoside triphosphate can be labeled.Examples of possible labels include, but are not limited to aradioisotope, an enzyme, an enzyme cofactor, an enzyme substrate, anenzyme inhibitor, a dye, a hapten, a chemiluminescent molecule, afluorescent molecule, a phosphorescent molecule, anelectrochemiluminescent molecule, a chromophore, a magnetic particle, anaffinity label, a chromogenic agent, an azide group or other groups usedfor click chemistry, and other moieties known in the art.

In one aspect, the nucleoside triphosphates synthesized according to themethods of the invention can be used for synthesizing nucleic acidmolecules. Nucleic acid in the context of the present invention includesbut is not limited to deoxyribonucleic acid (DNA), ribonucleic acid(RNA) and peptide nucleic acid (PNA). A nucleic acid of the inventionalso includes artificial genetic polymers, commonly referred to as XNAsor ‘xeno-nucleic acids’ where the backbone structure contains a sugarother than ribose or deoxyribose. While some of these molecules can beconsidered natural derivatives of RNA, like arabino nucleic acid (ANA),threose nucleic acid (TNA), and glycerol nucleic acid (GNA), others arecompletely unnatural, like locked nucleic acid (LNA), cyclohexenenucleic acid (CeNA), and hexitol nucleic acid (HNA).

Therefore in one embodiment, the invention provides artificial orsynthetic nucleic acid molecules which incorporate one or more naturalor modified nucleoside triphosphate of the invention. The length of thenucleic acids may vary. The nucleic acids may be modified, e.g. maycomprise one or more modified nucleobases or modified sugar moieties(e.g., comprising methoxy groups). The backbone of the nucleic acid maycomprise one or more peptide bonds as in peptide nucleic acid (PNA). Thenucleic acid may comprise a base analog such as non-purine ornon-pyrimidine analog or nucleotide analog. It may also compriseadditional attachments such as proteins, peptides and/or or amino acids.

Activated Nucleoside Monophosphates

In one embodiment, the invention relates to the synthesis of activatednucleoside monophosphates for use in synthesizing a nucleosidetriphosphate of the invention. An activated nucleoside monophosphatesfor use in the methods of the invention can be synthesized from anynucleoside monophosphate. In various embodiments, a nucleosidemonophosphate is a naturally occurring nucleoside monophosphate, anunnatural nucleoside monophosphate or a modified nucleosidemonophosphate.

In one embodiment, the activated nucleoside monophosphate comprises2-methylimidazole linked to a phosphate group. In one embodiment, thephosphate group is linked to a sugar moiety which is attached to anitrogenous base. In one embodiment, the sugar of the activatednucleoside monophosphate comprises ribose. In one embodiment, the sugarof the activated nucleoside monophosphate comprises deoxyribose.

In one embodiment, the activated nucleoside monophosphate is representedby formula (5)

wherein R₅ is selected from the group of hydrogen, aryl, and heteroaryl;and R₆ is a nitrogenous base. In one embodiment, the nitrogenous base isa natural nitrogenous base or artificial nitrogenous base. In oneembodiment, R₅ is phenyl.

A variety of purines, purine analogs, pyrimidines, pyrimidine analogs,and other heterocycles as nitrogenous bases have been well documented(Chemistry of Nucleosides and Nucleotides Vol 1, 2, 3, edited byTownsend, Plenum Press, 1988, 1991, 1994). The condensations of sugarswith nitrogenous bases to yield nucleosides are frequently usedreactions in nucleoside chemistry. Well-established procedures andmethodologies can be found in the literature (Vorbruggen et al., Chem.Ber. 1981, 114, 1234-1268, 1279-1286; Wilson et al., Synthesis, 1995,1465-1479).

A large number of known nucleosides are prepared from the modificationsof purine and pyrimidine nucleosides. The modifications can be done onthe sugars and/or nucleoside bases. A simple, widely-used reaction isthe nucleophilic substitution of halopurine or halopyrimidine base by avariety of nucleophiles such as hydroxide, ammonia, hydrogen sulfide,alkoxides, amines, alkylthiol, hydrazine, hydroxyamines, azide, cyanide,and hydride. This type of reactions can be very useful for preparationof 2-substituted purine nucleoside, 6-substituted purine nucleosides,8-substituted purine nucleosides, 2,6-disubstituted purine nucleosides,2,8-disubstituted purine nucleosides, 6,8-disubstituted purinenucleosides, 2,6,8-trisubstituted purine nucleosides (Halbfinger et al.,J. Med. Chem. 1999, 42, 5323-5337, Lin et al., J. Med. Chem. 1985, 28,1481-1485; Bressi et al., J. Med. Chem. 2000, 43, 4135-4150). Thesesubstitution reactions are readily extended to purine nucleoside analogssuch as 7-deazapurine nucleosides, 7-deaza-8-azapurine nucleosides,8-azapurine nucleosides, 3-deazapurine nucleosides, 3-deaza-8-azapurinenucleosides, and 3,8-dideazapurine nucleosides. For instance, a numberof 7-deaza-7-substituted purine nucleoside have been prepared throughsuch substitutions (Ugarkar et al., J. Med. Chem. 2000, 43, 2894-2905).The same methodologies can be used for the preparation of 4-substitutedpyrimidine nucleosides, 5-substituted pyrimidine nucleosides,4,5-disubstituted pyrimidine nucleosides, 5-substituted 6-azapyrimidinenucleosides, 5-substituted 6-azapyrimidine nucleosides, and4,5-disubstituted 6-azapyrimidine nucleosides.

The sugar moieties of synthesized nucleosides can be further modified.There are a variety of reactions which can be used to modify the sugarmoiety of nucleosides. The reactions frequently used includedeoxygenation, oxidation/addition, substitution, and halogenation. Thedeoxygenations are useful for the preparation of 2′-deoxy-, 3′-deoxy-,and 2′,3′-dideoxy-nucleosides. A widely-used reagent is phenylchlorothionoformate, which reacts with the hydroxy of nucleosides toyield a thionocarbonate. The treatment of the thionocarbonate withtributyltin hydride and AIBN yields deoxygenated nucleosides. Theoxidation/addition includes the conversion of a hydroxy group to acarbonyl group, followed by a nucleophilic addition, resulting inC-alkylated nucleosides and C-substituted nucleosides. The substitutionmay be just a simple displacement of a hydroxyl proton by alkyl, or maybe a conversion of a hydroxyl to a leaving group, followed by anucleophilic substitution. The leaving group is usually a halogen,mesylate, tosylate, nisylate, or a triflate. A variety of nucleophilescan be used, resulting in 2-, or 3-substituted nucleosides. Halogenationcan be used to prepare 1′-halo-, 2′-halo-, 3′-halo-, or4′-halonucleosides. Chlorination and fluorination are commonly used andresult in important fluoro-sugar and chloro-sugar nucleosides.

Nucleosides that can be included in an activated nucleosidemonophosphate according to the methods of the invention include, but arenot limited to, adenosine, cytidine, guanosine, uridine,2′-deoxyadenosine, 2′-deoxycytidine, 2′-deoxyguanosine, thymidine,inosine, 9-(β-D-arabinofuranosyl)adenine,1-(β-D-arabinofuranosyl)cytosine, 9-(β-D-arabinofuranosyl)guanine,1-(β-D-arabinofuranosyl)uracil, 9-(β-D-arabinofuranosyl)hypoxanthine,1-(β-D-arabinofuranosyl)thymine, 3′-azido-3′-deoxythymidine,3′-azido-2′, 3′-dideoxyuridine, 3′-azido-2′, 3′-dideoxycytidine,3′-azido-2′, 3′-dideoxyadenosine, 3′-azido-2′, 3′-dideoxyguanosine,3′-azido-2′, 3′-dideoxyinosine, 3′-deoxythymidine, 2′,3′-dideoxyuridine, 2′, 3′-dideoxyinosine, 2′, 3′-dideoxyadenosine, 2′,3′-dideoxycytidine, 2′, 3′-dideoxyguanosine, 9-(2,3-dideoxy-1-β-D-ribofuranosyl)-2, 6-diaminopurine, 3′-deoxy-2′,3′-didehydrothymidine, 2′, 3′-didehydro-2′, 3′-dideoxyuridine, 2′,3′-didehydro-2′, 3′-dideoxycytidine, 2′, 3′-didehydro-2′,3′-dideoxyadenosine, 2′, 3′-didehydro-2′, 3′-dideoxyguanosine, 2′,3′-didehydro-2′, 3′-dideoxyinosine, 3-deazaadenosine, 3-deazaguanosine,3-deazainosine, 7-deazaadenosine, 7-deazaguanosine, 7-deazainosine,6-azauridine, 6-azathymidine, 6-azacytidine, 5-azacytidine,9-(β-D-ribofuranosyl)-6-thiopurine,6-methylthio-9-(β-D-ribofuranosyl)purine,2-amino-9-(β-D-ribofuranosyl)-6-thiopurine,2-amino-6-methylthio-9-(β-D-ribofuranosyl)purine, 5-fluorocytidine,5-iodocytidine, 5-bromocytidine, 5-chlorocytidine, 5-fluorouridine,5-iodouridine, 5-bromouridine, 5-chlorouridine, 2′-C-methyladenosine,2′-C-methylcytidine, 2′-C-methylguanosine, 2′-C-methylinosine,2′-C-methyluridine, 2′-C-methylthymidine, 2′-deoxy-2′-fluoroadenosine,2′-deoxy-2′-fluorocytidine, 2′-deoxy-2′-fluoroguanosine,2′-deoxy-2′-fluorouridine, 2′-deoxy-2′-fluoroinosine,2′-α-fluorothymidine, 2′-deoxy-2′-fluoroarabinoadenosine,2′-deoxy-2′-fluoroarabinocytidine, 2′-deoxy-2′-fluoroarabinoguanosine,2′-deoxy-2′-fluoroarabinouridine, 2′-deoxy-2′-fluoroarabinoinosine,2′-β-fluorothymidine, 2′-O-methyladenosine, 2′-O-methylcytidine,2′-O-methylguanosine, 2′-O-methylinosine, 2′-O-5-dimethyluridine,2′-C-ethynylcytidine, 2′-C-ethynylguanosine, 2′-C-ethynyluridine,2′-C-ethynylinosine, 2′-C-ethynyl-5-methyluridine,3′-C-ethynyladenosine, 3′-C-ethynylcytidine, 3′-C-ethynylguanosine,3′-C-ethynyluridine, 3′-C-ethynylinosine, 3′-C-ethynyl-5-methyluridine,3′-deoxyadenosine, 3′-deoxycytidine, 3′-deoxyguanosine, 3′-deoxyuridine,3′-deoxyinosine, 4′-C-ethynyladenosine, 4′-C-ethynylcytidine,4′-C-ethynylguanosine, 4′-C-ethynyluridine, 4′-C-ethynylinosine,4′-C-ethynylthymidine, 4′-C-methyladenosine, 4′-C-methylcytidine,4′-C-methylguanosine, 4′-C-methyluridine, 4′-C-methylinosine,4′-C-methylthymidine, 2′-C-methyl-7-deazaadenosine,2′-C-methyl-7-deazaguanosine, 2′-C-methyl-3-deazaadenosine,2′-C-methyl-3-deazaguanosine, 2′-O-methyl-7-deazaadenosine,2′-O-methyl-7-deazaguanosine, 2′-O-methyl-3-deazaadenosine,2′-O-methyl-3-deazaguanosine, 2′-C-methyl-6-azauridine,2′-C-methyl-5-fluorouridine, 2′-C-methyl-5-fluorocytidine,2′-C-methyl-2-chloroadenosine, 2′-deoxy-7-deazaadenosine,2′-deoxy-3-deazaadenosine, 2′-deoxy-7-deazaguanosine,2′-deoxy-3-deazaguanosine, 2′-deoxy-6-azauridine,2′-deoxy-5-fluorouridine, 2′-deoxy-5-fluorocytidine,2′-deoxy-5-iodouridine, 2′-deoxy-5-iodocytidine,2′-deoxy-2-chloroadenosine, 2′-deoxy-2-fluoroadenosine,3′-deoxy-7-deazaadenosine, 3′-deoxy-7-deazaguanosine,3′-deoxy-3-deazaadenosine, 3′-deoxy-3-deazaguanosine,3′-deoxy-6-azauridine, 3′-deoxy-5-fluorouridine, 3′-deoxy-5-iodouridine,3′-deoxy-5-fluorocytidine, 3′-deoxy-2-chloroadenosine, 2′,3′-dideoxy-7-deazaadenosine, 2′, 3′-dideoxy-7-deazaguanosine, 2′,3′-dideoxy-3-deazaadenosine, 2′, 3′-dideoxy-3-deazaguanosine, 2′,3′-dideoxy-6-azauridine, 2′, 3′-dideoxy-5-fluorouridine, 2′,3′-dideoxy-5-fluorouridine, 2′, 3′-dideoxy-5-iodocytidine, 2′,3′-dideoxy-2-chloroadenosine, 2′, 3′-dideoxy-β-L-cytidine, 2′,3′-dideoxy-β-L-adenosine, 2′, 3′-dideoxy-β-L-guanosine,3′-deoxy-β-L-thymidine, 2′, 3′-dideoxy-5-fluoro-β-L-cytidine,3-L-thymidine, 2′-deoxy-β-L-cytidine, 2′-deoxy-β-L-adenosine,2′-deoxy-β-L-guanosine, 2′-deoxy-β-L-inosine, β-L-cytidine,j3-L-adenosine, β-L-guano sine, β-L-uridine, β-L-inosine, 2′,3′-didehydro-2′, 3′-dideoxy-β-L-cytidine, 2′,3′-didehydro-3′-dideoxy-β-L-thymidine, 2′, 3′-didehydro-2′,3′-dideoxy-β-L-adenosine, 2′, 3′-didehydro-2′, 3′-dideoxy-β-L-guanosine,2′, 3′-didehydro-2′, 3′-dideoxy-3-L-5-fluorocytidine, 2′-deoxy-2′,2′-difluorocytidine, 9-(β-D-arabinofuranosyl)-2-fluoroadenine,2′-deoxy-2′(E)-fluoromethylenecytidine, 2′-deoxy-2′(Z)-fluoromethylenecytidine, (−)-2′, 3′-dideoxy-3′-thiacytidine, (+)-2′,3′-dideoxy-3′-thiacytidine, 1-β-D-ribofuranosyl-1, 2,4-triazole-3-carboxamide, 1-β-L-ribofuranosyl-1, 2,4-triazole-3-carboxamide, 1-β-D-ribofuranosyl-1, 3-imidazolium-5-olate,1-β-L-ribofuranosyl-1, 3-imidazolium-5-olate,1-β-D-ribofuranosyl-5-ethynylimidazole-4-carboxamide,1-β-L-ribofuranosyl-5-ethynylimidazole-4-carboxamide,1-(2-deoxy-2-fluoro-(β-D-arabinofuranosyl)-5-iodouracil,1-(2-deoxy-2-fluoro-(β-D-arabinofuranosyl)-5-iodocytosine,1-(2-deoxy-2-fluoro-β-L-arabinofuranosyl)-5-methyluracil,1-β-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil,E-5-(2-bromovinyl)-2′-deoxyuridine, 5-trifluoromethylthymidine,1-β-D-arabinoifuranosyl-5-propynyluracil,1-(2-deoxy-2-fluoro-1-β-D-arabinofuranosyl)-5-ethyluracil, 2′,3′-dideoxy-3′-fluoroguanosine, 3′-deoxy-3′-fluorothymidine, (+)-(1α, 2β,3α)-9-[2, 3-bis(hydroxymethyl)-1-cyclobutyl]adenine, (+)-(1α, 2β,3α)-9-[2, 3-bis(hydroxymethyl)-1-cyclobutyl]guanine, (+)-(1β, 2α,3β)-9-[2, 3-bis(hydroxymethyl)-1-cyclobutyl]guanine, (+)-(1β, 2α,3β)-9-[2, 3-bis(hydroxymethyl)-1-cyclobutyl]adenine, (1R, 3S,4R)-9-(3-hydroxy-4-hydroxymethylcyclopent-1-yl)guanine, (1 S, 2R,4R)-9-(1-hydroxy-2-hydroxymethylcyclopent-4-yl)guanine, (2R,4R)-9-(2-hydroxymethyl-1, 3-dioxolan-4-yl)-2, 6-diaminopurine, (2R,4R)-1-(2-hydroxymethyl-1, 3-dioxolan-4-yl)cytosine, (2R,4R)-9-(2-hydroxymethyl-1, 3-dioxolan-4-yl)guanine, (2R,4R)-1-(2-hydroxymethyl-1, 3-dioxolan-4-yl)-5-fluorocytosine, (1R, 2S,4S)-9-(4-hydroxy-3-hydroxymethyl-2-methylenecyclopent-4-yl]guanine, and(1 S, 3R,4S)-9-(3-hydroxy-4-hydroxymethyl-5-methylenecyclopent-1-yl]guanine.

Methods

In one aspect, the invention provides methods of synthesizingphosphorylated molecules. In one aspect, the method comprises forming amixture comprising an activated precursor molecule and an organicmolecule comprising at least one phosphate moiety of the invention andincubating the mixture in the presence of a catalyst to catalyzesynthesis of a phosphorylated molecule. In various embodiments, thecompositions and methods of the invention can be used to synthesizemolecules with mono-, di-, tri-, tetra-, penta- or hexa-peptidemoieties. The methods disclosed herein can be used to synthesizephosphorylated sugar molecules, phosphorylated proteins or peptides,phosphorylated lipids, or phosphorylated nucleic acid molecules.

In one aspect, the invention provides methods of synthesizing natural ormodified nucleoside monophosphates including, but not limited to, 5′nucleoside monophosphate (NMP), 2′-deoxynucleoside-5′-monophosphate(dNMP), and analogs thereof. In one aspect, the method comprises forminga mixture comprising an activated nucleoside molecule and an organicmolecule comprising a monophosphate moiety of the invention andincubating the mixture in the presence of a catalyst to catalyzesynthesis of a nucleoside monophosphate.

In one aspect, the invention provides methods of synthesizing natural ormodified nucleoside diphosphates including, but not limited to, 5′nucleoside diphosphate (NDP) and 2′-deoxynucleoside-5′-diphosphates(dNDP), and analogs thereof. In one aspect, the method comprises forminga mixture comprising an activated nucleoside monophosphate and anorganic molecule comprising a monophosphate moiety of the invention andincubating the mixture in the presence of a catalyst to catalyzesynthesis of a nucleoside diphosphate. In one aspect, the methodcomprises forming a mixture comprising an activated nucleoside moleculeand an organic molecule comprising a pyrophosphate moiety of theinvention and incubating the mixture in the presence of a catalyst tocatalyze synthesis of a nucleoside diphosphate.

In one aspect, the invention provides methods of synthesizing natural ormodified nucleoside triphosphates including, but not limited to, 5′nucleoside triphosphate (NTP) and 2′-deoxynucleoside-5′-triphosphates(dNTP), and analogs thereof. In one aspect, the method comprises forminga mixture comprising an activated nucleoside monophosphate and anorganic molecule comprising a pyrophosphate moiety of the invention andincubating the mixture in the presence of a catalyst to catalyzesynthesis of a nucleoside triphosphate. In one aspect, the methodcomprises forming a mixture comprising an activated nucleoside moleculeand an organic molecule comprising a triphosphate moiety of theinvention and incubating the mixture in the presence of a catalyst tocatalyze synthesis of a nucleoside triphosphate.

In one aspect, the invention provides methods of synthesizing natural ormodified nucleoside tetraphosphates including, but not limited to, 5′nucleoside tetraphosphate (Np₄) and2′-deoxynucleoside-5′-tetraphosphates (dNp₄), and analogs thereof. Inone aspect, the method comprises forming a mixture comprising anactivated nucleoside monophosphate and an organic molecule comprising atriphosphate moiety of the invention and incubating the mixture in thepresence of a catalyst to catalyze synthesis of a nucleosidetetraphosphate. In one aspect, the method comprises forming a mixturecomprising an activated nucleoside molecule and an organic moleculecomprising a tetraphosphate moiety of the invention and incubating themixture in the presence of a catalyst to catalyze synthesis of anucleoside tetraphosphate. In one aspect, the method comprises forming amixture comprising an activated nucleoside diphosphate molecule and anorganic molecule comprising a pyrophosphate moiety of the invention andincubating the mixture in the presence of a catalyst to catalyzesynthesis of a nucleoside tetraphosphate.

In one aspect, the invention provides methods of synthesizing natural ormodified nucleoside pentaphosphates including, but not limited to, 5′nucleoside pentaphosphate (Np₅) and2′-deoxynucleoside-5′-pentaphosphates (dNp₅), and analogs thereof. Inone aspect, the method comprises forming a mixture comprising anactivated nucleoside monophosphate and an organic molecule comprising apentaphosphate moiety of the invention and incubating the mixture in thepresence of a catalyst to catalyze synthesis of a nucleosidepentaphosphate. In one aspect, the method comprises forming a mixturecomprising an activated nucleoside molecule and an organic moleculecomprising a pentaphosphate moiety of the invention and incubating themixture in the presence of a catalyst to catalyze synthesis of anucleoside pentaphosphate. In one aspect, the method comprises forming amixture comprising an activated nucleoside triphosphate molecule and anorganic molecule comprising a pyrophosphate moiety of the invention andincubating the mixture in the presence of a catalyst to catalyzesynthesis of a nucleoside pentaphosphate. In one aspect, the methodcomprises forming a mixture comprising an activated nucleosidediphosphate molecule and an organic molecule comprising a triphosphatemoiety of the invention and incubating the mixture in the presence of acatalyst to catalyze synthesis of a nucleoside pentaphosphate.

In one aspect, the invention provides methods of synthesizing natural ormodified nucleoside hexaphosphates including, but not limited to, 5′nucleoside hexaphosphate (Np₆) and 2′-deoxynucleoside-5′-hexaphosphates(dNp₆), and analogs thereof. In one aspect, the method comprises forminga mixture comprising an activated nucleoside monophosphate and anorganic molecule comprising a pentaphosphate moiety of the invention andincubating the mixture in the presence of a catalyst to catalyzesynthesis of a nucleoside hexaphosphate. In one aspect, the methodcomprises forming a mixture comprising an activated nucleosidepentaphosphate and an organic molecule comprising a monophosphate moietyof the invention and incubating the mixture in the presence of acatalyst to catalyze synthesis of a nucleoside hexaphosphate. In oneaspect, the method comprises forming a mixture comprising an activatednucleoside tetraphosphate molecule and an organic molecule comprising apyrophosphate moiety of the invention and incubating the mixture in thepresence of a catalyst to catalyze synthesis of a nucleosidehexaphosphate. In one aspect, the method comprises forming a mixturecomprising an activated nucleoside diphosphate molecule and an organicmolecule comprising a tetraphosphate moiety of the invention andincubating the mixture in the presence of a catalyst to catalyzesynthesis of a nucleoside hexaphosphate. In one aspect, the methodcomprises forming a mixture comprising an activated nucleosidetriphosphate molecule and an organic molecule comprising a triphosphatemoiety of the invention and incubating the mixture in the presence of acatalyst to catalyze synthesis of a nucleoside hexaphosphate.

Similarly, the compositions and methods of the invention can be used tosynthesize molecules with heptapeptides, octapeptides, nonapeptides,decapeptides or longer peptide moieties.

Although it is not intended that the method of the invention be limitedto a particular set of reaction conditions, in one embodiment, thereaction conditions include a first step of contacting an activatednucleoside monophosphate with an organic molecule comprising a phosphatemoiety of the invention in the presence of an acid catalyst at roomtemperature (rt) to form a reaction intermediate, followed by secondstep of contacting the reaction intermediate with a base at atemperature of 37° C.

Although it is not intended that the method of the invention be limitedto any particular acid catalyst, in one embodiment, any acidic compound(including the so-called Lewis acids) may be used as catalysts. In oneembodiment, the acid catalyst is ZnCl₂, H₂SO₄, HCl, H₃PO₄ or BF₃. Inanother embodiment, the acid catalyst is a sulfonic acid or its salt,comprising ortho-toluenesulfonic acid, meta-toluenesulfonic acid,alkylbenzenesulfonic acid, secondary alkyl-sulfonic acid, sulfonicresin, alkylsulfate, alkylbenzenesulfonate, alkyl-sulfonate andsulfosuccinic acid. In a preferred embodiment, the acid catalyst isZnCl₂.

Although it is not intended that the method of the invention be limitedto a particular base, in one embodiment, the base is selected from agroup of organic or inorganic basic materials comprising the alkalimetal bases such as alkali metal hydroxide, carbonates, andbicarbonates. In another embodiment, the base is selected from a groupcomprising the alkaline earth bases such as calcium oxide and magnesiumoxide. In another embodiment, aluminum bases such as aluminum hydroxideor its basic alkali aluminum components are contemplated. In a. furtherembodiment, the base is selected from a group comprising ammonia-basedcompounds, such as ammonium hydroxide, and amines including, but notlimited to, primary, secondary tertiary and heterocyclic amines.

Applications

The compositions comprising modified nucleotides and methods catalyzingthe incorporation of modified nucleotides of the present invention maybe used in a wide variety of protocols and technologies. For example, incertain embodiments, the modified nucleotides are used in the fields ofmolecular biology, genomics, transcriptomics, epigenetics, nucleic acidsequencing, and the like. That is, modified nucleotides may be used inany technology that may require or benefit from specific incorporationof modified nucleotides. Exemplary technologies, include, but are notlimited to cDNA library construction; DNA epigenome and RNA methylomeassays, high-throughput next generation sequencing technologiesincluding but not limited to Illumina, SOLiD, and Ion Torrentsequencing; and single nucleic acid molecule real time sequencing (SMRT)including, but not limited to, technologies from Pacific Bioscience andOxford Nanopore Technologies such as zero-mode waveguide or nanoporesequencing, respectively.

Pharmaceutical Compositions

In some embodiments, nucleoside triphosphates can function aspharmaceutical agents, e.g. 3′-azido-3′-deoxythymidine (AZT, an anti-HIVdrug) triphosphate and arabinosylcytosine (Cytarabine, an anticancerdrug) triphosphate. In addition, nucleotides have been considered asantimetabolite drugs, agents for the treatment of diseases and disordersassociated with infection (e.g., U.S. Pat. No. 5,763,447). Nucleosidetriphosphates or nucleoside triphosphate analogs have also beendescribed for the treatment of diseases and disorders including, but notlimited to, sinusitis (e.g., U.S. Pat. No. 5,789,391), ostitis media(e.g., U.S. Pat. No. 6,423,694), inflammatory conditions (e.g., U.S.Patent Application No. 2005/261239), and cancer (e.g., U.S. Pat. No.5,049,372).

Therefore, in one embodiment, the invention also relates to apharmaceutical composition comprising a therapeutically effective amountof a synthesized nucleotide of the invention, a pharmaceuticallyacceptable salt thereof, optionally in combination with one or moreother active ingredients and/or with a pharmaceutically acceptablecarrier. Moreover, the above any of the compounds may be used in amethod for the treatment of a disease or disorder including, but notlimited to, a microbial infection or proliferative disorder, comprisingadministering a therapeutically effective amount of any of the abovecompounds to a subject in need thereof.

The pharmaceutical composition of the present invention comprises atleast one nucleotide triphosphate synthesized according to the methodsof the invention, or pharmaceutically acceptable salts, esters orprodrugs thereof as active ingredients. The compositions include thosesuitable for oral, topical, intravenous, subcutaneous, nasal, ocular,pulmonary, and rectal administration. The compounds of the invention canbe administered to mammalian individuals, including humans, astherapeutic agents.

For example, the compounds of the invention are useful as antiviralagents. The present invention provides a method for the treatment of apatient afflicted with a viral infection comprising administering to thepatient a therapeutically effective antiviral amount of a compound ofthe invention. The term “viral infection” as used herein refers to anabnormal state or condition characterized by viral transformation ofcells, viral replication and proliferation. Viral infections for whichtreatment with a compound of the invention will be particularly usefulinclude the viruses mentioned above.

A “therapeutically effective amount” of a compound of the inventionrefers to an amount which is effective, upon single or multiple doseadministration to the patient, in controlling the growth of e.g., themicrobe or tumor or in prolonging the survivability of the patientbeyond that expected in the absence of such treatment. As used herein,“controlling the growth” refers to slowing, interrupting, arresting orstopping the microbial or proliferative transformation of cells or thereplication and proliferation of the microbe and does not necessarilyindicate a total elimination of e.g., the microbe or tumor.

Accordingly, the present invention includes pharmaceutical compositionscomprising, as an active ingredient, at least one of the compounds ofthe invention in association with a pharmaceutical carrier. Thecompounds of this invention can be administered by oral, parenteral(intramuscular, intraperitoneal, intravenous (IV) or subcutaneousinjection), topical, transdermal (either passively or usingiontophoresis or electroporation), transmucosal (e.g., nasal, vaginal,rectal, or sublingual) or pulmonary (e.g., via dry powder inhalation)routes of administration or using bioerodible inserts and can beformulated in dosage forms appropriate for each route of administration.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is admixed with at least one inert pharmaceutically acceptablecarrier such as sucrose, lactose, or starch. Such dosage forms can alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., lubricating, agents such as magnesium stearate. In thecase of capsules, tablets, and pills, the dosage forms may also comprisebuffering agents. Tablets and pills can additionally be prepared withenteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, with the elixirscontaining inert diluents commonly used in the art, such as water.Besides such inert diluents, compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring, and perfuming agents.

Preparations according to this invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdosage forms may also contain adjuvants such as preserving, wetting,emulsifying, and dispersing agents. They may be sterilized by, forexample, filtration through a bacteria retaining filter, byincorporating sterilizing agents into the compositions, by irradiatingthe compositions, or by heating the compositions. They can also bemanufactured using sterile water, or some other sterile injectablemedium, immediately before use.

Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance,excipients such as cocoa butter or a suppository wax. Compositions fornasal or sublingual administration are also prepared with standardexcipients well known in the art.

Topical formulations will generally comprise ointments, creams, lotions,gels or solutions. Ointments will contain a conventional ointment baseselected from the four recognized classes: oleaginous bases;emulsifiable bases; emulsion bases; and water-soluble bases. Lotions arepreparations to be applied to the skin or mucosal surface withoutfriction, and are typically liquid or semiliquid preparations in whichsolid particles, including the active agent, are present in a water oralcohol base. Lotions are usually suspensions of solids, and preferably,for the present purpose, comprise a liquid oily emulsion of theoil-in-water type. Creams, as known in the art, are viscous liquid orsemisolid emulsions, either oil-in-water or water-in-oil. Topicalformulations may also be in the form of a gel, i.e., a semisolid,suspension-type system, or in the form of a solution.

Formulations of these drugs in dry powder form for delivery by a drypowder inhaler offers yet another means of administration. Thisovercomes many of the disadvantages of the oral and intravenous routes.

The dosage of active ingredient in the compositions of this inventionmay be varied; however, it is necessary that the amount of the activeingredient shall be such that a suitable dosage form is obtained. Theselected dosage depends upon the desired therapeutic effect, on theroute of administration, and on the duration of the treatment desired.Generally, dosage levels of between 0.001 to 10 mg/kg of body weightdaily are administered to mammals.

Kits

The present invention also relates to a kit for performing any of theabove described methods, wherein the kit comprises a synthesizednucleoside triphosphate or a nucleic acid molecule comprising asynthesized nucleoside triphosphate. In one embodiment, the kit maycomprise a mixture of synthesized nucleotides. In some embodiments, oneor more of the components are premixed in the same reaction container.

In particular embodiments, the kit additionally comprises instructionalmaterial.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless so specified. Thus, the invention should in no way be construedas being limited to the following examples, but rather, should beconstrued to encompass any and all variations which become evident as aresult of the teaching provided herein. Without further description, itis believed that one of ordinary skill in the art can, using thepreceding description and the following illustrative examples, make andutilize the compounds of the present invention and practice the claimedmethods. The following working examples therefore, specifically pointout the preferred embodiments of the present invention, and are not tobe construed as limiting in any way the remainder of the disclosure

Example 1: Synthesis of Natural and Modified Nucleoside Triphosphates

Examples for the synthesis of the nucleotides of the present inventionare given in this section.

Synthesis of Activated Thymidine Monophosphate

Activated thymidine monophosphate was synthesized according to thescheme shown in FIG. 10.

Synthesis of p-Toluene Pyrophosphate

A p-toluene pyrophosphate reagent was synthesized according to thescheme shown in FIG. 8.

Synthesis of Pyrene Pyrophosphate

A pyrene pyrophosphate reagent was synthesized according to the schemeshown in FIG. 9.

Synthesis of Nucleoside Triphosphates

5′-thymidine triphosphate was synthesized according to the scheme shownin FIG. 2B. The activated nucleoside monophosphate is reacted with anorganic pyrophosphate reagent (FIG. 1, FIG. 2A, FIG. 5) to form anintermediate product (FIG. 2C) which is then reduced to form anucleoside triphosphate. The reaction shown in FIG. 2B is catalyzed byZnCl₂.

The reaction of FIG. 2B results in the formation of dinucleosidediphosphate, a minor contaminant (FIG. 4).

5′-thymidine triphosphate (5′-TTP), 3′-thymidine triphosphate (3′-TTP),and α-L-threofuranosyl 3′-thymidine triphosphate (tTTP)) as well as fournaturally occurring nucleoside triphosphates have all been synthesizedusing the methods of the invention (FIG. 6 and FIG. 7).

Example 2: P(V) Reagents for the Scalable Synthesis of Natural andModified Nucleoside Triphosphates

Historically, the synthesis of chemically modified nucleosidetriphosphates has been limited by such factors as scalability, lowyields, difficult reaction conditions, and tedious purificationsprotocols (Burgess and Cook, 2000, Chem. Rev. 100, 2047-2060; Kore andSrinivasan, 2013, Curr. Org. Syn. 10, 903-934; Hollenstein, 2012,Molecules 17, 13569-13591). Efforts to overcome these problems haveresulted in an astonishing number of publications, nearly all of whichrequire HPLC purification (Burgess and Cook, 2000, Chem. Rev. 100,2047-2060; Kore and Srinivasan, 2013, Curr. Org. Syn. 10, 903-934;Hollenstein, 2012, Molecules 17, 13569-13591). Attempts to synthesizeα-L-threofuranosyl adenosine triphosphates using the classic Yoshikawaor Ludwig-Eckstein methods (Yoshikawa et al., 1967, Tetrahedron Lett. 8,5065-5068; Ludwig and Eckstein, 1989, J. Org. Chem. 54, 631-635)produced complex mixtures of phosphorylated compounds with the desiredcompound present as a minor product (FIG. 11).

Therefore, a fundamentally different approach was developed for thesynthesis of natural and modified nucleoside triphosphates that requiredinvention of a novel P(V)-based organic pyrophosphate reagent to mediateP—O bond formation between the α- and β-phosphate positions. Here, asynthetic route to nucleic acid building blocks is described that (1) isnot constrained to natural substrates; (2) is scalable to gramquantities of material; (3) eliminates the requirement for HPLCpurification; and (4) avoids the need for freeze drying. Thepyrophosphate reagent and activated nucleoside monophosphates describedin this study are readily produced on scales of 5-10 grams usingsynthetic methodology that is inexpensive, straightforward and highyielding. The availability of these reagents makes it possible togenerate 1.0 gram of nucleoside triphosphate, which is enough materialto perform 5 liters of PCR or 100,000 PCR reactions with a standard 50μL volume. Unlike conventional tributyl ammonium pyrophosphate, pyrenepyrophosphate is a solid at room temperature and stable to atmosphericconditions, allowing it to be used without specialized equipment andstored for long periods of time without loss of activity. Last, thepyrene pyrophosphate method was found to be superior for synthesizingchemically modified nucleotides that were challenging to synthesizeusing the classic Yoshikawa or Ludwig-Eckstein methods (14, 15). Basedon these considerations, the current approach provides an alternativeparadigm for synthesizing large quantities of nucleoside triphosphatesfor emerging applications in biotechnology and molecular medicine.

The invention establishes an Hoard-Ott-like procedure (FIG. 12) usingnucleoside and pyrophosphate reagents that were suitably hydrophobic soas to generate nucleoside triphosphate derivatives that were amenable topurification by silica gel chromatography but could be readily convertedto the desired compound using standard deprotection and precipitationconditions (Hoard and Ott, 1965, J. Am. Chem. Soc. 87, 1785-1788). Thistype of convergent synthesis strategy provides a direct and scalableroute to natural and chemically modified nucleoside triphosphates usingoperationally simple protocols that process and discovery chemists wouldfind appealing. Critical to this effort was the need to establish a newclass of organic pyrophosphate reagents that contain a large hydrophobicmoiety attached to the pyrophosphate group via a cleavable linker and todemonstrate that these reagents could mediate the synthesis ofnucleoside triphosphates. Reducing this concept to practice involved asystematic evaluation of three key components: (1) hydrophobic groupsnecessary to construct a stable organic pyrophosphate reagent that is asolid compound at room temperature and exhibits minimal hygroscopicproperties; (2) a strong leaving group to activate the nucleosidemonophosphate for nucleophilic attack by the pyrophosphate reagent; and(3) a suitable Lewis acid to accelerate phosphodiester bond formation(FIG. 12). The goals of this study were ultimately met through asystematic exploration and subsequent optimization of reactionconditions which revealed that pyrene, 2-methylimidazole, and zincchloride provided the optimal hydrophobic moiety, leaving group, andLewis acid catalyst, respectively, necessary to mediate the synthesis ofnucleoside triphosphates in their fully protected form. The crystalstructure of the pyrene pyrophosphate reagent verified correct synthesisof the organic pyrophosphate reagent (FIG. 13).

The materials and methods used are now described.

Analytical Reverse-Phase HPLC Analysis. 2 μL of reaction crude(compounds: 10-11a-d, and 16-17, and 22-23a-d, and 28-29) in DMF or 1 μLof purified pyrene substituted nucleoside triphosphates (compounds:12a-d, and 18, and 24a-d, and 30) in methanol or 3 μL of nucleosidetriphosphates (compounds: 13a-d, and 19, and 25a-d, and 31) in H₂O wasadded to 50 L of 0.1 M triethylammonium acetate (TEAA) buffer pH 7.0.The solution was centrifuged by 4000 rpm for 2 minutes at roomtemperature and 30 μL of supernatant was injected for HPLC analysis.Reaction progress of the synthesis of nucleoside monophosphates(compounds: 10a-d, and 16, and 22a-d, and 28), nucleosidephosphor-2-methylimidazolides (compounds: 11a-d, and 17, and 23a-d, and29), pyrene substituted nucleoside triphosphates (compounds: 12a-d, and18, and 24a-d, and 30) and nucleoside triphosphates (compounds: 13a-d,and 19, and 25a-d, and 31) in FIG. 32, FIG. 44, FIG. 56, FIG. 68, FIG.80, FIG. 92, FIG. 101, FIG. 110, FIG. 119, and FIG. 128 was monitored byanalytical HPLC chromatography with the gradient from 0% to 50% ofacetonitrile in 0.1 M triethylammonium acetate buffer pH 7.0 over fortyminutes. Purity determination of synthesized 5′-dNTPs (compounds: 13a-d)was monitored by the coinjection with the commercial 5′-dNTPs purchasedfrom Sigma-Aldrich by analytical HPLC chromatography in with thegradient from 0% to 10% of acetonitrile in 0.1 M triethylammoniumacetate buffer pH 7.0 over forty minutes of analytical run.

PCR Fidelity Assay.

A 100 μl PCR reaction was performed in 1× ThermoPol buffer (20 mMTris-HCl, 10 mM (NH₄)₂SO₄, 10 mM KCl, 2 mM MgSO₄, 0.1% Triton X-100, pH8.8, NEB), 0.5 LpM of Fwd [CAACCGGTCCCCACGTTGCC] (SEQ ID NO:1) and Rev[AACGGCTGGGAGAACCTGGTTCTCAATGTA] (SEQ ID NO:2) PCR primers, 400 μM dNTPs(Chemically Synthesized vs Life Technologies), 4.4 ng of pGDR11 KOD-RSplasmid [Target sequence:CAACCGGTCCCCACGTTGCCGTTGCCAAGAGGTTGGCCGCGAGAGGAGTCAAAATACGCCCTGGAACGGTGATAAGCTACATCGTGCTCAAGGGCTCTGGGAGGATAGGCGACAGGGCGATACCGTTCGACGAGTTCGACCCGACGAAGCACAAGTACGACGCCGAGTACTACATTGAGAACCAGGTTCTCCCAGCCGT T] (SEQ ID NO:3) withfinal concentration of 5 units/100 μL Taq polymerase (NEB). The PCRconditions were: 95° C., 2.5 min (melt), 95° C., 30 sec, 62° C., 45 sec,72° C., 30 sec for 20 cycles and an additional 72° C., 2 min. Theamplified amplicon (200 bp) was agarose purified, ligated into a TOPO-TAvector, and subsequently cloned into NEB DH5α E. coli competent cellsfollowing the manufacturer's instructions. Individual colonies weregrown in liquid media and sequenced using the MβF primer by Retrogen,San Diego, Calif. DNA sequences were aligned and analyzed using MEGA7software. Five sequences clones were analyzed for each condition to givea total of 750 nucleotide positions.

TNA Transcription Assay.

Primer-extension reactions were performed in a final volume of 20 μl.

Each reaction contained 10 pmol of primer[IR680-5′-GTCCCCTTGGGGATACCACC-3′] (SEQ ID NO:4) annealed to 10 pmol oftemplate [5′-ATCGAGTACAGTCAGATCGATATGATCTATATATTAATTAGGTGGTATCCCCAAGGGGAC-3′] (SEQ ID NO:5), 1× ThermoPol buffer, 0.5 μM KOD-RS, 100 μMof each tNTP. Reactions were incubated for 120 min at 55° C., quenchedwith stop buffer (40% Formamide and 1×TBE buffer, 10 mM EDTA), andanalyzed by 10% denaturing urea PAGE.

The results of the experiments are now described.

The pyrene pyrophosphate method was applied to the synthesis of naturalthymidine-5′-triphosphate (dTTP). As shown in FIG. 14A-FIG. 14C,activated thymidine-5′-monophosphate is readily converted to a fullyprotected thymidine-5′-triphosphate derivative, which was purified bysilica gel chromatography, deprotected, and precipitated to obtain thedesired product as a highly pure compound in sodium form. In thisreaction, 10 molar equivalents of ZnCl₂ enabled a near quantitativecoupling (>95%) of the pyrene pyrophosphate reagent to the activatednucleoside monophosphate after 3 hours of stirring in DMF at 24° C. Thephosphorylation reaction also generated trace amounts (1-2%) of unwantedside products, including dinucleotide diphosphate, that were removed bysilica gel purification using a 10% H₂O/isopropanol mobile phasecontaining 1% Hünig's base [diisopropylethylamine, DIPEA] as an organiccounterion. Following purification, the triphosphate intermediate wasdeprotected with concentrated ammonium hydroxide (33% aq.) andprecipitated as the sodium salt using standard conditions. AnalyticalHPLC analysis reveals that chemically synthesized dTTP was equivalent inpurity to a commercial standard (FIG. 14C), demonstrating that theprocess of the nucleotide triphosphate synthesis is capable ofgenerating material that is identical in purity to commercial compoundsobtained by conventional enzymatic synthesis.

Because enzymatic approaches are not compatible with most nucleic acidanalogs, it was decided to broaden the scope of the reaction by applyingthe pyrene pyrophosphate method to a select panel of thymidine analogs(FIG. 15). The list of target molecules includedthymidine-3′-triphosphate (a regioisomer of natural dTTP),α-L-threofuranosyl thymidine 3′-triphosphate (tTTP, a building block forthreose nucleic acid, TNA) (Schoning et al., 2000, Science 290,1347-1351), and L-thymidine-5′-triphosphate (the mirror image form ofnatural D-dTTP). The last two examples have become valuable compoundsfor biomedical research due to recent advances in polymerase engineeringthat have enabled the synthesis of artificial genetic polymers withnon-natural sugar-phosphate backbones (Larsen et al., 2016, Nat. Commun.7, 11235; Wang et al., 2016, Nat. Chem. 8, 698-704), some of which havebeen used to evolve biologically stable aptamers and catalysts (Pinheiroet al., 2012, Science 336, 341-344; Yu et al., 2012, Nat. Chem. 4,183-187; Taylor et al., 2015, Nature 518, 427-430; Wang et al., 2018,Nat. Commun., in press). As with natural dTTP, each unnatural thymidinetriphosphate was obtained in highly pure form (>95%) as the desiredsodium salt. Occasionally, a small amount of nucleoside diphosphate(dNDP) (˜1-2%) was observed after deprotection of the protectednucleoside triphosphates. dNDPs are common contaminants of commercialnucleotide triphosphates due to the slow hydrolysis of ATP to ADP(Hulett, 1970, Nature 225, 1248-1249). However, these molecules do notinterfere with normal DNA synthesis as natural DNA polymerases arehighly specific for dNTP substrates (Steitz et al., 1994, Science 266,2022-2025).

Next, a complete set of DNA nucleoside triphosphates (dNTPs) wasprepared using the pyrene pyrophosphate method. Each activatednucleoside monophosphate was converted to the desired nucleosidetriphosphate following the standard procedure of pyrene pyrophosphatecoupling, purification by silica gel chromatography, deprotection withconcentrated ammonium hydroxide, and precipitation as the sodium salt(FIG. 16A). Concurrently, all four TNA nucleoside triphosphates (tNTPs)were synthesized, which are the substrates for engineered TNApolymerases developed by directed evolution (FIG. 16B) (Larsen et al.,2016, Nat. Commun. 7, 11235). In all cases, analytical HPLCchromatograms reveal high conversion (>95%) of activated nucleosidetriphosphate into the fully protected nucleoside triphosphate. Followingdeprotection and precipitation, the desired DNA and TNA nucleosidetriphosphates were produced in highly pure form on scales (>500milligrams) and timeframes (2-3 days) vastly exceeding those oftraditional protocols.

Since nucleoside triphosphates would ultimately be used as substratesfor oligonucleotide synthesis, the use of these reagents wasinvestigated in a conventional DNA synthesis assay. One criticalquestion that this study aimed to address is whether chemicallysynthesized DNA triphosphates function with same level of efficiency asenzymatically produced dNTPs obtained from commercial venders. Toaddress this question, the polymerase chain reaction (PCR) was used toamplify an arbitrary DNA sample in reaction mixtures that containedeither chemically synthesized or commercial dNTPs. A 200 base pairsegment was amplified that defines the finger subdomain of an archaealDNA polymerase isolated from the thermophilic species Thermococcuskodakarensis (Kod) (Chim et al., 2017, Nat Commun 8, 1810). Analysis ofthe resulting PCR amplicons by agarose gel electrophoresis confirmedthat chemically synthesized dNTP substrates function identically tocommercial dNTPs, as both reactions produce equivalent amounts of DNA ateach cycle of PCR amplification (FIG. 16A). Moreover, DNA sequencing ofthe amplified product failed to identify any instances of insertions,deletions, or mutations, indicating that the reactions proceed with hightemplate-sequence fidelity.

Next, the set of chemically synthesized TNA triphosphates was evaluatedas substrates for TNA synthesis using an engineered TNA polymerase (Chimet al., 2017, Nat Commun 8, 1810). In this assay, a DNA primer-templatecomplex was extended with TNA using either newly synthesized tNTPs ortNTPs obtained by a traditional Hoard-Ott-like approach that requiresHPLC purification (Bala et al., 2017, J. Org. Chem. 82, 5910-5916).Analysis of the resulting primer-extension reactions by denaturingpolyacrylamide gel electrophoresis reveals that the two sets of TNAsubstrates generate equivalent amounts of full-length TNA product after3 hours of incubation at 55° C. (FIG. 16B). This result, along with thePCR assay, confirms that the pyrene pyrophosphate method produces highquality nucleoside triphosphates that function as substrates for naturaland engineered polymerases.

Example 3: Synthesis of 1-(2-(pyrenesulfonyl)ethyl)pyrophosphate

FIG. 17 shows the synthesis scheme for1-(2-(pyrenesulfonyl)ethyl)pyrophosphate (compound 7). FIG. 18 throughFIG. 31 provide spectra of the synthesized intermediates and product.

Methods used for the synthesis are now described.

2-(Pyrenethio)ethanol (Compound 2)

To a solution containing 9.98 g (177.8 mmol) of potassium hydroxide in250 mL of anhydrous DMF was added 12.5 mL (177.8 mmol) of2-mercaptoethanol. The reaction mixture was heated to 80° C. withstirring until potassium hydroxide dissolved. A premade solutioncontaining 25 g (88.92 mmol) of 1-bromopyrene (compound 1) in 200 mL ofanhydrous DMF was dropwise added to the reaction and stirred at 110° C.for 3 hours. Then, the reaction was cooled to room temperature,condensed to 100 mL of solution under diminished pressure and dilutedinto 500 mL of CH₂Cl₂. The organic layer was washed with 200 mL of H₂Ofour times. And the combined aqueous layer was back extracted with 200mL of CH₂Cl₂ two times. The organic layers were combined and evaporatedto the dryness under diminished pressure. The crude product was purifiedby silica column chromatography with eluents (CH₂Cl₂/Hexane, 25% to 50%,then EtOAc/CH₂Cl₂, from β% to 2%) to afford the compound 2 as ayellowish solid; yield: 21.3 g (85.8%); silica gel TLC (EtOAc/Hexane,1:2) R_(f)=0.35; ¹H NMR (400 MHz, CDCl₃) δ 8.61 (d, 1H, J=9.2 Hz),8.07-8.00 (m, 4H), 7.93-7.81 (m, 4H), 3.71 (t, 2H, J=6.0 Hz), 3.18 (t,2H, J=6.0 Hz); HRMS (ESI-TOF) calcd. for C₁₈H₁₄OSNa [M+Na]⁺ 301.0663;found 301.0658. Compound 2 has been reported in Kathayat et al., 2013,J. Am. Chem. Soc. 135, 12612-12614.

2-(Pyrenesulfonyl)ethanol (Compound 3)

To a solution containing 71 g (114.9 mmol, 80% technical grade) ofmagnesium bis(monoperoxyphthalate) hexahydrate in 450 mL of anhydrousDMF was slowly added a pre-made solution of 21.3 g (76.6 mmol) ofcompound (2) in 150 mL of anhydrous DMF at β° C. The reaction wasstirring at room temperature for 12 hours. At which the TLC showed thereaction was finished, the reaction was dropwise added to a solutioncontaining 2 L of satd. NaHCO_(3(aq)) while stirring. The precipitatewas washed with 300 mL of H₂O twice, and dried under high vacuum toafford the compound 3 as a yellowish solid; yield: 22.8 g (96%); silicagel TLC (EtOAc/CH₂Cl₂, 1:20) R_(f)=0.22; ¹H NMR (400 MHz, CDCl₃) δ 9.00(d, 1H, J=9.2 Hz), 8.71 (d, 1H, J=8.4 Hz), 8.34-8.31 (m, 3H), 8.27-8.23(m, 2H), 8.14-8.09 (m, 2H), 4.00 (t, 2H, J=5.2 Hz), 3.64 (t, 2H, J=5.2Hz); ¹³C NMR (125.8 MHz, CDCl₃-10% DMSO-d₆) δ 135.5, 130.8, 130.7,130.6, 130.3, 129.9, 128.9, 127.5, 127.2, 127.1, 127.0, 126.9, 125.0,124.1, 123.9, 122.4, 58.6, 56.1; HRMS (ESI-TOF) calcd. for C₁₈H₁₄O₃SNa[M+Na]⁺ 333.0561; found 333.0571.

Dibenzyl-1-(2-(pyrenesulfonyl)ethyl) monophosphate (Compound 4)

To a mixture containing 5 g (16.13 mmol) of 2-(pyrenesulfonyl)ethanol(compound 3), 2.03 g (29.03 mmol) of tetrazole in 106 mL of anhydroussolution (MeCN/CH₂Cl₂, 1:1) was slowly added 6.64 mL (20.96 mmol) of(BnO)₂PN(i-Pr)₂ at room temperature. The reaction was sonicated untilthe solid disappeared (otherwise add additional 20 mL of anhydrous DMF)and the mixture was stirring at room temperature for 3 hours. Thenreaction was cooled to −40° C. followed by adding 15 mL of H₂O₂ (33% inH₂O) for additional 1 h stirring at room temperature. The solution wascondensed to 20 mL under diminished pressure and poured to 150 mL EtOAc.The organic layer was washed with 100 mL brine, 200 mL satd. NaHCO₃, 200mL H₂O, dried over MgSO₄ and evaporated under diminished pressure. Thecrude product was purified by silica column chromatography with eluent(CH₂Cl₂/Hexane, from 25% to 100%, then MeOH/CH₂Cl₂, from 1% to 3%) toafford the yellowish solid product, compound 4; yield: 6.73 g (73.2%);TLC (EtOAc/CH₂Cl₂, 1:20) R_(f)=0.34; ¹H NMR (400 MHz, CDCl₃) δ 8.89 (d,1H, J=9.2 Hz), 8.60 (d, 1H, J=8.0 Hz), 8.15-8.11 (m, 3H), 8.01-7.94 (m,3H), 7.82 (d, 1H, J=8.8 Hz), 7.20-7.16 (m, 6H), 7.06-7.04 (m, 4H), 4.67(d, 4H, J=8.4 Hz), 4.36 (dd, 2H, J=14, 6.0 Hz), 3.70 (t, 2H, J=6.0 Hz);¹³C NMR (125.8 MHz, CDCl₃) δ 135.2, 135.2, 135.1, 130.5, 130.4, 129.9,129.5, 128.7, 128.3, 128.3, 127.6, 127.4, 127.1, 127.0, 126.7, 126.6,124.5, 123.9, 123.4, 122.0, 69.2 (d, J C,P=5.8 Hz), 60.7 (d, J C,P=5.0Hz), 56.3 (d, J C,P=7.2 Hz); ³¹P NMR (162 MHz, CDCl₃) δ −0.41; HRMS(ESI-TOF) calcd. for C₃₂H₂₇O₆PSNa [M+Na]⁺ 593.1164; found 593.1158.

1-(2-(Pyrenesulfonyl)ethyl) monophosphate (Compound 5)

To a solution containing 6.73 g (11.8 mmol) ofdibenzyl-1-(2-(pyrenesulfonyl)ethyl) monophosphate (compound 4) in 50 mLof MeOH was purged with nitrogen gas. The solution was added to 0.1-0.2mass equivalent of 10% Pd/C, and repurged with nitrogen gas followed byadding hydrogen gas. The mixture was stirred at room temperature for 3hours with monitoring by TLC (MeOH/CH₂Cl₂, 1:10 with 1% Et₃N). Theresulting suspension was filtered over a pad of celite, washed with 100mL of MeOH four times, evaporated, and then coevaporated with dryC₂H₄Cl₂ under reduced pressure to afford the product, compound 5, as ayellowish solid; yield 4.42 g (96%); TLC (MeOH/CH₂Cl₂, 1:10 with 1%triethylamine) R_(f)=0; ¹H NMR (400 MHz, CD₃OD) δ 8.72 (d, 1H, J=9.6Hz), 8.44 (d, 1H, J=8.4 Hz), 8.00-7.90 (m, 4H), 7.79-7.75 (m, 2H), 7.66(d, 1H, J=8.8 Hz), 4.34 (dd, 2H, J=12.4, 6.0 Hz), 3.82 (t, 2H, J=6.0Hz); ¹³C NMR (125.8 MHz, CD₃OD+10% DMSO-d₆) δ 136.6, 131.7, 131.5,131.5, 131.4, 130.8, 129.8, 128.4, 128.2, 128.0, 127.8, 127.7, 125.5,125.2, 124.4, 123.2, 60.9 (d, J C,P=4.2 Hz), 57.6 (d, J C,P=7.2 Hz); ³¹PNMR (162 MHz, CD₃OD) δ −0.56. HRMS (ESI-TOF) calcd. for C₁₈H₁₄O₆PS[M−H]⁻ 389.0249; found 389.0234.

1-(2-(Pyrenesulfonyl)ethyl)-(β-dibenzyl)pyrophosphate (Compound 6)

To the 4.42 g (11.3 mmol) of 1-(2-(pyrenesulfonyl)ethyl) monophosphate(compound 5) in 39.3 mL of anhydrous dichloromethane was added 5.33 mL(15.82 mmol) of (BnO)₂PN(i-Pr)₂ at room temperature under a nitrogenatmosphere. After two hours stirring, the reaction was slowly added to6.16 mL (33.9 mmol) of tert-butyl hydrogen peroxide (5.5 M in decane) at−40° C. and stirred for an additional 30 minutes at room temperature.The reaction was evaporated under diminished pressure and the crudeproduct was purified by silica column chromatography with the eluents(MeCN then MeOH/CH₂Cl₂ from 1% to 3% containing 1% triethylamine) toafford the solid product, compound 6; yield: 6.5 g (76.6%); TLC (2:100MeOH—CH₂Cl₂ with 1% triethylamine) R_(f)=0.26; ¹H NMR (400 MHz, CD₃OD) δ8.90 (d, 1H, J=9.6 Hz), 8.56 (d, 1H, J=8.0 Hz), 8.15-8.11 (m, 3H),8.06-8.00 (m, 2H), 7.94 (t, 1H, J=8.0 Hz), 7.87 (d, 1H, J=8.8 Hz), 7.19(m, 10H), 4.94 (d, 4H, J=7.6 Hz), 4.32 (dd, 2H, J=15.2, 6.8 Hz), 3.75(t, 2H, J=6.8 Hz); ¹³C NMR (125.8 MHz, CDCl₃) δ 136.0, 135.9, 135.6,130.9, 130.7, 130.7, 130.6, 130.0, 129.1, 128.5, 128.3, 127.8, 127.7,127.3, 127.2, 127.1, 127.0, 125.1, 124.3, 124.0, 122.6, 69.3 (d, JC,P=5.5 Hz), 59.7 (d, J C,P=5.5 Hz), 57.0 (d, J C,P=6.9 Hz); ³¹P NMR(162 MHz, CDCl₃) δ −11.2 (d, J=18.5 Hz), −11.6 (d, J=18.5 Hz); HRMS(ESI-TOF) calcd. for C₃₂H₂₇O₉P₂SNa₂ [M−H+2Na]⁺ 695.0646; found 695.0660.

1-2-(Pyrenesulfonyl)ethyl)pyrophosphate (Compound 7)

To a solution containing 6.5 g of1-(2-(pyrenesulfonyl)ethyl)-(β-dibenzyl)pyrophosphate (compound 6) in 30mL of MeOH was purged with nitrogen gas. The solution was added to0.1-0.2 mass equivalent of 10% Pd/C and purged with nitrogen gasfollowed by adding hydrogen gas. The mixture was stirred at roomtemperature for 3 h with monitoring by TLC (MeOH/CH₂Cl₂, 1:10 with 1%Et₃N). The resulting suspension was filtered over a pad of celite,washed with 100 mL of MeOH three times, evaporated, and coevaporatedwith dry C₂H₄Cl₂ under reduced pressure to afford the product as ayellowish solid (compound 7); yield: 4.27 g (90.9%); TLC (MeOH/CH₂Cl₂,1:10 with 1% triethylamine) R_(f)=0; ¹H NMR (400 MHz, CD₃OD) δ 8.93 (d,1H, J=9.2 Hz), 8.61 (d, 1H, J=8.0 Hz), 8.29-8.22 (m, 4H), 8.13 (d, 1H,J=8.8 Hz), 8.05-8.00 (m, 2H), 4.35 (m, 2H), 3.91 (t, 2H, J=10.4 Hz); ¹³CNMR (125.8 MHz, D₂O) 135.0, 130.7, 130.1, 129.7, 129.2, 129.0, 128.3,127.3, 127.1, 127.0, 126.6, 126.3, 124.4, 123.6, 122.4, 121.8, 60.6,57.3 (d, J C,P=6.0 Hz); ³¹P NMR (162 MHz, D₂O) δ −10.0 (d, J=16.8 Hz),−11.5 (d, J=16.8 Hz); HRMS (ESI-TOF) calcd. for C₁₈H₁₄O₉P₂SNa₃[M−2H+3Na]⁺ 536.9527; found 536.9521.

Example 4: Synthesis of Nucleoside Triphosphates

FIG. 32 through FIG. 141 depict schematic diagrams for the synthesis ofnucleoside triphosphates and NMR spectra of the synthesizedintermediates and products.

Methods used for the synthesis are now described.

General Procedure A: Synthesis of Protected Nucleoside Monophosphates.

To a mixture containing suitably protected free nucleoside (compounds:8a-d, 14, 20a-d, and and 1.8 equivalents of tetrazole in an anhydroussolvent (MeCN/CH₂Cl₂, 1:1) was added to 1.3 equivalents of(BnO)₂PN(i-Pr)₂ under a nitrogen atmosphere. The mixture was stirred atroom temperature for 1-3 hours and the reaction was monitored by TLC. Atwhich the starting material was consumed, the reaction was slowly addedexcess H₂O₂ (33% in H₂O) at −40° C. and the resulting mixture wasstirred at room temperature for 1 hour. The solution was then dilutedwith 15-20 times volume of CH₂Cl₂ and the organic layer was sequentiallywashed with saturated NaHCO_(3(aq)), brine, and water. The organicextracts were combined, dried with MgSO₄, and evaporated underdiminished pressure. The crude residue was purified by silica gelchromatography and the fractions containing the product were collectedand evaporated to afford the product, compounds: 9a-d, 15, 21a-d, or 27.

General Procedure B: Synthesis of Nucleoside Monophosphates.

To a solution containing the protected nucleoside monophosphate(compounds 9a-d, 15, 21a-d, and in MeOH was purged with nitrogen gas.The solution was added 0.1-0.2 mass equivalent of 10% Pd/C, andre-purged with nitrogen gas followed by adding hydrogen gas. The mixturewas stirred at room temperature for 3-5 hours with monitoring by TLC(MeOH/CH₂Cl₂, 1:10 with 1% triethylamine). Then the solution was purgedwith nitrogen, filtered over a pad of celite, and washed with MeOH orMeOH containing 2% triethylamine. The filtrate was collected, evaporatedand coevaporated with dry 1, 2-dichloroethane under reduced pressure toafford the product, compounds: 10a-d, 16, 22a-d, or 28.

General Procedure C: Synthesis of Activated Nucleoside Monophosphates.

To a solution containing the nucleoside monophosphate (compounds 10a-d,16, 22a-d and 28) in anhydrous DMF under a nitrogen atmosphere wasslowly added 5 equivalents of anhydrous triethylamine at β° C. After 5minutes with stirring, 2 equivalents of 2-methylimidazole was addedfollowed by 2 equivalents of triphenylphosphine. After 10 minutes ofstirring at room temperature, 2 equivalents of 2, 2′-dipyridyl disulfidewas added and stirring was continued for an additional 3 hours at roomtemperature with monitoring by analytical HPLC (mobile phase: MeCN/0.1 MTEAA buffer, from β% to 50% over 40 minutes). After consumption of thestarting material, the product was precipitated by adding the reactiondropwise with stirring to 300 mL of diethyl ether. The precipitate wascollected by centrifuging at 4400 rpm for 15 minutes at roomtemperature. The supernatant was discarded, and the pellet wasresuspended with minimal amount of CH₂Cl₂ or anhydrous DMF (DMF is usedwhen the nucleoside monophosphate is not soluble in CH₂Cl₂). Thesolution was added dropwise to a premade solution of ether/ethylacetate/triethylamine (5:10:1) containing 8 equivalents of sodiumperchlorate for a second precipitation. The suspended solid wascentrifuged at 4400 rpm for 15 minutes at room temperature, thesupernatant was discarded, and the pellet was washed twice with 40 mL ofmixed solvent (ether/ethyl acetate, 1:2), and dried under high vacuum toafford the product, compounds: 11a-d, 17, 23a-d or 29.

General Procedure D: Synthesis of Fully Protected NucleosideTriphosphates.

To a mixture containing the activated nucleoside monophosphate(compounds 11a-d, 17, 23a-d, and 29) and 1.2 equivalents of1-(2-(pyrenesulfonyl)ethyl)pyrophosphate (compound 7) was added alongwith 8-10 equivalents of a premade solution of ZnCl₂ (1.0 M in anhydrousDMF) under a nitrogen atmosphere. The mixture was stirred at roomtemperature for 3-5 hours and the reaction progress was monitored byHPLC (MeCN/0.1 M TEAA buffer, from β% to 50% over 40 minutes). At whichthe starting material was consumed, the reaction was dropwise added toethyl acetate or ether for precipitation. The precipitate wascentrifuged and collected at 4400 rpm for 10 minutes at roomtemperature, and the supernatant was discarded. The pellet wasresuspended by 20% H₂O in MeCN with 2% Hunig's base and the solid wasfiltered by pyrex glass funnel with filter paper. The filtrate wascollected and evaporated under diminished pressure and dry-packingloaded to silica gel for normal phase silica column chromatography. Thefractions containing the product were collected and evaporated underdiminished pressure at 30-40° C. The product was resuspended with CH₂Cl₂and insoluble silica gel was removed by filtration. The filtrate wascollected and evaporated to dryness to afford the product, compounds:12a-d, 18, 24a-d, or 30.

General Procedure E: Synthesis of Free Nucleoside Triphosphates.

To a solution of fully protected nucleoside triphosphate (compounds:12a, 18, 24a, and 30) in 50 mL of 33% NH₄OH_((aq)) Was stirred for 18hours at room temperature or (compounds: 12b-d, and 24b-d) was stirredfor 3 hours at 37° C. and 15 hours at room temperature in a sealed tube.After the reaction, the solvent was evaporated under diminishedpressure. The solid was resuspended with MilliQ water and the aqueoussolution was washed with CH₂Cl₂ and ethyl acetate. The organic portionwas discarded and the aqueous extract was collected, and evaporatedunder diminished pressure. The crude solid was resuspended with minimalamount of RNAse free water, filtrated by 0.22 um syringe filter anddropwise added to the forty times volume of acetone at room temperaturecontaining 15 equivalents of sodium perchlorate. The resultingsuspension was centrifuged at 4400 rpm for 15 minutes at roomtemperature. The supernatant was discarded and the pellet was washedwith organic solution (acetone/CH₂Cl₂, 10:1) twice to afford theproduct, compounds: 13a-d, 19, 25a-d, or 31.

Synthesis of thymidine-5′-triphosphate

FIG. 32 shows the synthesis scheme for1-(2-(pyrenesulfonyl)ethyl)pyrophosphate (dTTP, compound 13a). FIG. 33through FIG. 43 provide spectra of the synthesized intermediates andproduct.

Methods used for the synthesis are now described.

3′-O-Benzoyl-2′-deoxythymidine-5′-dibenzylmonophosphate (Compound 9a)

General procedure A with 1 g (2.89 mmol) of3′-O-benzoyl-2′-deoxythymidine compound 8a, 364.4 mg (5.2 mmol) oftetrazole, 28.9 mL of anhydrous solution (MeCN/CH₂Cl₂, 1:1), 1.3 mL(3.76 mmol) of dibenzyl-N, N-diisopropylphosphoramidite for 3 hoursreaction at room temperature. Then, 6 mL of 30% H₂O_(2(aq)) for 1 houroxidation reaction at room temperature. Column chromatography witheluents (MeOH/CH₂Cl₂, from 1% to 1.4%) to afford the product compound 9aas a white solid; yield: 1.2 g (68.5%); TLC (MeOH/CH₂Cl₂, 1:40)R_(f)=0.23; ¹H NMR (400 MHz, CD₃OD) δ 8.03-8.00 (m, 2H), 7.62-7.58 (m,1H), 7.48-7.45 (m, 3H), 7.36-7.29 (m, 10H), 6.32 (dd, 1H, J=8.4, 6.0Hz), 5.42-5.40 (m, 1H), 5.12-5.07 (m, 4H), 4.34-4.28 (m, 3H), 2.47 (ddd,1H, J=14.4, 6.0, 2.0 Hz), 2.26-2.18 (m, 1H), 1.77 (d, 3H, J=1.2 Hz);HRMS (ESI-TOF) calcd. for C₃₁H₃₁N₂O₉PNa [M+Na]⁺ 629.1665; found629.1664. Compound 9a has been reported in De et al., 2014, Eur. J. Org.Chem. 2322-2348.

3′-O-Benzoyl-2′-deoxythymidine-5′-monophosphate (Compound 10a)

General procedure B with 1.2 g (1.98 mmol) of compound 9a, 50 mL ofMeOH, and 200 mg of 10% Pd/C for 3 hours stirring at room temperature.The suspension was filtered over a pad of celite, and washed with 100 mLof MeOH containing 2% triethylamine four times to afford the productcompound 10a as a white foam of triethylammonium salt; yield: 1.11 g(88.7%); TLC (MeOH/CH₂Cl₂, 1:10 with 1% triethylamine) R_(f)=0; ¹H NMR(400 MHz, DMSO-d₆) δ 11.41 (s, 1H), 8.09 (d, 2H, J=8.0 Hz), 7.77-7.74(m, 2H), 7.62 (t, 2H, J=7.6 Hz), 6.41 (dd, 1H, J=8.0, 6.8 Hz), 5.57 (s,1H), 4.41 (s, 1H), 4.18 (m, 2H), 2.58-2.57 (m, 2H), 1.88 (s, 3H); ¹³CNMR(125.8 MHz, DMSO-d₆) 168.6, 167.3, 152.7, 138.6, 135.6, 131.0, 130.4,130.3, 113.0, 86.2, 85.1 (d, J C,P=8.7 Hz), 78.0, 66.2, 37.9, 31.9,13.2; ³¹P NMR (162 MHz, DMSO-d₆) δ 0.24; HRMS (ESI-TOF) calcd. forC₁₇H₁₈N₂O₉PNa₂ [M−H+2Na]⁺ 471.0545; found 471.0539.

3′-O-Benzoyl-2′-deoxythymidine-5′-phosphor-2-methylimidazolide (Compound11a)

General procedure C with 1.11 g (1.76 mmol) of compound 10a, 7.8 mL ofanhydrous DMF, 1.63 mL (11.72 mmol) of triethylamine, 385 mg (4.69 mmol)of 2-methylimidazole, 1.23 g (4.69 mmol) of triphenylphosphine, 1.04 g(4.72 mmol) of dipyridyl disulfide for 2 hours reaction at roomtemperature. First precipitation was achieved with 250 mL of diethylether. The product was resuspended with 10 mL of DMF and dropwise addedto the solution containing 2.01 g of sodium perchlorate, 15 mL oftriethylamine in 300 mL of ethyl acetate for second precipitation. Theproduct was afforded as a white solid compound 11a; yield: 0.89 g(98.9%); ³¹P NMR (162 MHz, D₂O) δ −6.83; HRMS (ESI-TOF) calcd. forC₂₁H₂₃N₄O₈PNa [M+Na]⁺ 513.1151; found 513.1141.

3′-O-Benzoyl-2′-deoxythymidine-5′-(γ-(2-(pyrenesulfonyl)ethyl))triphosphate(Compound 12a)

General procedure D with 0.89 g (1.74 mmol) of compound 11a, 1.09 g(1.91 mmol) of 1-2-(pyrenesulfonyl)ethyl) pyrophosphate (compound 7) and13.92 mL (13.92 mmol) of ZnCl₂ solution (1.0 M in anhydrous DMF) for 3hours stirring at room temperature. Then crude was precipitated bydropwise adding the solution to the 300 mL of ethyl acetate withstirring. After centrifugation, the pellet was resuspended by 20%H₂O/MeCN containing 2% Hunig's base and filtered by pyrex glass funnel.The filtrate was evaporated and the crude material was purified bysilica column chromatography with eluents (H₂O/(isopropanol-MeCN 1:1)from 2% to 7% containing 1% diisopropylethylamine (DIPEA) to obtain purecompound 12a; yield: 1.43 g (64.9%); TLC (H₂O/acetone 1:10 with 2%diisopropylethylamine) Rf=0.29; ′H NMR (400 MHz, D₂O) δ 8.25 (d, 1H,J=8.8 Hz), 8.10 (d, 1H, J=7.6 Hz), 7.41 (d, 1H, J=9.2 Hz), 7.33-7.27 (m,3H), 7.19-7.13 (m, 4H), 6.94-6.89 (m, 3H), 6.68 (t, 2H, J=6.4 Hz), 5.61(s, 1H), 5.16 (s, 1H), 4.49 (s, 2H), 4.15-4.07 (m, 2H), 3.78 (m, 3H)2.04-1.94 (m, 2H), 1.70 (s, 3H); ³¹P NMR (162 MHz, D₂O) δ −10.22 (brs,2P), −20.50 (brs, 1P); HRMS (ESI-TOF) calcd. for C₃₅H₃₁N₂O₁₇P₃SNa₃[M−2H+3Na]⁺ 945.0250; found 945.0261.

2′-deoxythymidine-5′-triphosphate (Compound 13a)

General procedure E with 1.43 g (1.13 mmol) of compound 12a, 50 mL of33% NH₄OH_((aq)) for 18 h stirring at room temperature. The productcompound 13a was afforded as a white solid; yield: 453 mg (83.2%, T,ε₂₆₇: 9600 M⁻¹cm⁻¹); ′H NMR (400 MHz, D₂O) δ 7.74 (s, 1H), 6.37 (t, 1H,J=6.0 Hz), 4.69 (s, 1H), 4.28-4.24 (m, 3H), 2.42 (s, 2H), 1.96 (s, 3H);³¹PNMR (162 MHz, D₂O) δ −4.06 (d, J=16.2 Hz), −9.30 (d, J=16.2 Hz),−17.95 (brs).

Synthesis of 2′-deoxycytidine-5′-triphosphate

FIG. 44 shows the synthesis scheme for 2′-deoxycytidine-5′-triphosphate(dCTP, compound βb). FIG. 45 through FIG. 55 provide NMR spectra of thesynthesized intermediates and product.

Methods used for the synthesis are now described.

3′-O, N⁴-Dibenzoyl-2′-deoxycytidine-5′-dibenzylmonophosphate (Compound9b)

General procedure A with 1.04 g (2.40 mmol) of 3′-O,N⁴-dibenzoyl-2′-deoxythymidine compound 8b, 306 mg (4.31 mmol) oftetrazole, 16 mL of anhydrous solution (MeCN/CH₂Cl₂, 1:1), 0.99 mL (3.12mmol) of dibenzyl-N, N-diisopropylphosphoramidite for 1 hour stirring atroom temperature followed by 6 mL of H₂O₂ for 1 hour oxidation reaction.Column chromatography with eluents (MeOH/CH₂Cl₂, from β% to 12.5%) toafford the product compound 9b as a white solid; yield: 1.26 g (75.5%);TLC (MeOH/CH₂Cl₂, 1:60) R_(f)=0.32; ¹H NMR (400 MHz, CDCl₃) δ 8.12 (d,1H, J=7.6 Hz), 8.02 (d, 2H, J=7.6 Hz), 7.96 (d, 2H, J=7.6 Hz), 7.59-7.52(m, 2H), 7.48-7.41 (m, 5H), 7.36-7.28 (m, 10H), 6.37 (dd, 1H, J=8.4, 5.6Hz), 5.41 (d, 1H, J=6.4 Hz), 5.14-5.02 (m, 4H), 4.42-4.32 (m, 3H), 2.82(dd, 1H, J=14.0, 5.2 Hz), 2.05-1.97 (m, 1H); ¹³C NMR (125.8 MHz, CDCl₃)δ 165.7, 162.4, 154.6, 143.8, 135.4, 135.3, 133.4, 133.1, 132.9, 129.6,128.9, 128.7, 128.6, 128.6, 128.4, 127.9, 127.9, 127.7, 96.9, 87.0, 83.6(d, J C,P=7.8 Hz), 75.0, 69.7 (d, J C,P=5.3 Hz), 66.8 (d, J C,P=5.2 Hz),38.7; ³¹P NMR (162 MHz, CDCl₃) δ 0.76; HRMS (ESI-TOF) calcd. forC₃₇H₃₄N₃O₉PNa [M+Na]⁺ 718.1931; found 718.1949.

3′-O, N⁴-Dibenzoyl-2′-deoxycytidine-5′-monophosphate (Compound 10b)

General procedure B with 1.26 g (1.81 mmol) of compound 9b, 100 mL ofMeOH, and 250 mg of 10% Pd/C for 3 h stirring. The suspension wasfiltered over a pad of celite, washed with 100 mL of MeOH containing 2%triethylamine four times to afford the product compound 10a as a whitefoam of triethylammonium salt; yield: 1.05 g (84.1%); TLC (MeOH/CH₂Cl₂,1:10) R_(f)=0; ¹H NMR (400 MHz, CD₃OD) δ 8.68 (d, 1H, J=6.8 Hz), 8.08(d, 2H, J=7.6 Hz), 7.98 (d, 2H, J=7.6 Hz), 7.71 (d, 1H, J=7.2 Hz),7.65-7.62 (m, 2H), 7.55-7.49 (m, 4H), 6.45 (t, 1H, J=6.0 Hz), 5.69 (d,1H, J=4.0 Hz), 4.51 (s, 1H), 4.22 (s, 1H), 2.78 (dd, 1H, J=13.6, 4.0Hz), 2.59-2.52 (m, 1H); ¹³CNMR (125.8 MHz, CD₃OD) δ 168.9, 167.2, 164.9,157.9, 146.9, 134.8, 134.5, 134.0, 131.0, 130.6, 129.8, 129.7, 129.1,99.1, 88.7, 86.5 (d, J C,P=8.4 Hz), 77.8, 65.8, 40.1; ³¹P NMR (162 MHz,CD₃OD) δ 1.22; HRMS (ESI-TOF) calcd. for C₂₃H₂₂N₃O₉PNa [M+Na]⁺ 538.0991;found 538.0984.

3′-O, N⁴-Dibenzoyl-2′-deoxycytidine-5′-phosphor-2-methylimidazolide(compound 11b)

General procedure C with 1.05 g (1.46 mmol) of compound 10b, 7.3 mL ofanhydrous DMF, 1.02 mL (7.30 mmol) of triethylamine, 252 mg (3.07 mmol)of 2-methylimidazole, 918 mg (3.5 mmol) of triphenylphosphine, 804 mg(3.65 mmol) of dipyridyl disulfide for 2 hours reaction at roomtemperature. First precipitation was achieved with 250 mL of diethylether. The product was resuspended with 10 mL of DMF and dropwise addedto the solution containing 1.79 g of sodium perchlorate, 15 mL oftriethylamine in 300 mL of mixing solution (ethyl acetate/ether 1.5:1)for second precipitation. The product was afforded as a white solidcompound 11b; yield: 0.82 g (93.4%); ³¹P NMR (162 MHz, D₂O) δ −6.63;HRMS (ESI-TOF) calcd. for C₂₇H₂₅N₅O₈PNa₂ [M−H+2Na]⁺ 624.1236; found624.1256.

3′-O,N⁴-Dibenzoyl-2′-deoxycytidine-5′-(γ-(2-(pyrenesulfonyl)ethyl))triphosphate(Compound 12b)

General procedure D with 0.82 g (1.36 mmol) of compound 11b, 0.77 g(1.63 mmol) of 1-(2-(pyrenesulfonyl)ethyl) pyrophosphate (compound 7)and 13.6 mL (13.6 mmol) of ZnCl₂ solution (1.0 M in anhydrous DMF) for 3hours with stirring. Then crude was precipitated by dropwise adding thesolution to the 300 mL of ethyl acetate with stirring for precipitation.After centrifugation, the pellet was resuspended by 20% H₂O/MeCNcontaining 2% Hunig's base and filtered by pyrex glass funnel. Thefiltrate was evaporated and crude product was purified by silica columnchromatography with eluents 7% H₂O/isopropanol containing 1%diisopropylethylamine (DIPEA) and then H₂O in (isopropanol/MeCN 1:1)from 5% to 8% containing 1% diisopropylethylamine (DIPEA) to afford thewhite solid compound 12b; yield: 1.07 g (58.1%); TLC (H₂O/acetone 1:10containing 2% DIPEA): Rf=0.32; ′H NMR (500 MHz, D₂O/DMSO-d₆ 1:1) δ 11.15(s, 1H), 8.91 (s, 1H), 8.60-7.95 (m, 7H), 7.62-7.33 (m, 4H), 6.25 (s,1H), 5.47 (s, 1H), 4.33-3.90 (m, 4H), 2.34 (s, 1H); ³¹P NMR (162 MHz,D₂O/DMSO-d₆ 1:1) δ −12.13 (d, 1P, J=17.2 Hz), −12.38 (d, 1P, J=14.9 Hz),−22.59 (brs, 1P); HRMS (ESI-TOF) calcd. for C₄₁H₃₄N₃O₁₇P₃SNa₃[M−2H+3Na]⁺ 1034.0515; found 1034.0485.

2′-deoxythymidine-5′-triphosphate (compound βb)

General procedure E with 1.04 g (0.79 mmol) of compound 12b in 55 mL of33% NH₄OH_((aq)) for 3 hours deprotection at 37° C. and then 15 hoursdeprotection at room temperature. The product compound βb was affordedas a white solid; yield: 302 mg (82.5%, C, ε₂₈₀: 13100 M⁻¹cm⁻¹); ¹H NMR(400 MHz, D₂O) δ 7.96 (d, 1H, J=7.2 Hz), 6.36 (t, 1H, J=6.4 Hz), 6.18(t, 1H, J=6.8 Hz), 4.64 (s, 1H), 4.26 (s, 3H), 2.47-2.32 (m, 2H); ³¹PNMR (162 MHz, D₂O) δ −5.04 (brs), −9.23 (brs), −18.71 (brs).

Synthesis of 2′-deoxyadenosine-5′-triphosphate

FIG. 56 shows the synthesis scheme for 2′-deoxyadenosine-5′-triphosphate(dATP, compound βc). FIG. 57 through FIG. 67 provide NMR spectra of thesynthesized intermediates and product.

Methods used for the synthesis are now described.

3′-O, N⁶, N⁶-Tribenzoyl-2′-deoxyadenosine-5′-dibenzylmonophosphate(Compound 9c)

General procedure A with 1.03 g (1.83 mmol) of3′-O,N⁶,N⁶-tribenzoyl-2′-deoxyadenosine compound 8c, 233 mg (3.29 mmol)of tetrazole, 12 mL of anhydrous solution (MeCN/CH₂Cl₂, 1:1), 0.75 mL(2.38 mmol) of dibenzyl-N, N-diisopropylphosphoramidite for 1 hourstirring at room temperature. Then, the reaction was added to 6 mL ofH₂O₂ for 1 hour oxidation reaction. Column chromatography with eluents(EtOAc/hexane, from 33% to 66%) to afford the product compound 9c as awhite foam; yield: 1.33 g (88.3%); TLC (EtOAc/hexane, 1:60) R_(f)=0.32;¹H NMR (400 MHz, CDCl₃) δ 8.63 (s, 1H), 8.38 (s, 1H), 8.06 (d, 2H, J=8.0Hz), 7.87 (d, 3H, J=8.0 Hz), 7.64-7.61 (m, 1H), 7.50-7.46 (m, 4H),7.37-7.28 (m, 15H), 6.60-6.56 (m, 1H), 5.55 (d, 1H, J=5.6 Hz), 5.07-5.01(m, 4H), 4.40 (s, 1H), 4.34-4.24 (m, 2H), 2.72-2.67 (m, 2H); ¹³CNMR(125.8 MHz, CDCl₃) δ 172.7, 166.2, 153.2, 152.6, 152.3, 143.5, 134.5,134.1, 133.4, 129.9, 129.5, 129.1, 129.1, 129.1, 129.1, 129.1, 129.0,129.0, 128.6, 128.6, 128.1, 84.9, 83.9 (d, J C,P=10.4 Hz), 75.0, 69.7(d, J C,P=5.3 Hz), 66.8 (d, J C,P=5.2 Hz), 38.7; ³¹P NMR (162 MHz,CDCl₃) δ 0.70; HRMS (ESI-TOF) calcd. for C₄₅H₃₈N₅O₉PNa [M+Na]⁺ 846.2305;found 846.2291.

3′-O, N⁶, N⁶-Tribenzoyl-2′-deoxyadenosine-5′-monophosphate (Compound10c)

General procedure B with 1.33 g (1.62 mmol) of compound 9c, 100 mL ofMeOH, and 230 mg of 10% Pd/C for 3 hours stirring. The suspension wasfiltered over a pad of celite, washed with 100 mL of MeOH containing 2%triethylamine four times to afford the product compound 10c as a whitefoam of triethylammonium salt; yield: 0.88 g (84.5%); TLC (MeOH/CH₂Cl₂1:10 with 1% triethylamine) R_(f)=0; ¹H NMR (400 MHz, CD₃OD) δ 8.93 (s,1H), 8.69 (s, 1H), 8.06 (d, 2H, J=7.6 Hz), 7.80-7.78 (m, 3H), 7.62-7.58(m, 1H), 7.49-7.47 (m, 4H), 7.37-7.34 (m, 3H), 6.71 (t, 1H, J=1.6 Hz),5.77 (d, 1H, J=5.6 Hz), 4.47 (s, 1H), 4.17-4.13 (m, 2H), 3.14-3.12 (m,1H), 2.74 (dd, 1H, J=13.6, 5.6 Hz); ¹³C NMR (125.8 MHz, CD₃OD) δ173.7,167.2, 154.4, 153.1, 152.6, 146.3, 135.4, 134.5, 134.5, 134.2, 131.0,130.6, 130.4, 129.8, 129.7, 129.6, 129.4, 128.9, 86.0 (d, J C,P=10.4Hz), 77.9, 66.2, 55.6, 47.5, 39.1; ³¹P NMR (162 MHz, CD₃OD) δ 2.30; HRMS(ESI-TOF) calcd. for C₃₁H₂₅N₅O₉PNa₂ [M−H+2Na]⁺ 688.1185; found 688.1204.

3′-O, N⁶,N⁶-Tribenzoyl-2′-deoxyadenosine-5′-phosphor-2-methylimidazolide(Compound 11c)

General procedure C with 0.88 g (1.37 mmol) of compound 10c, 7.15 mL ofanhydrous DMF, 1.0 mL (7.19 mmol) of triethylamine, 239 mg (2.85 mmol)of 2-methylimidazole, 760 mg (2.90 mmol) of triphenylphosphine, 642 mg(2.92 mmol) of dipyridyl disulfide for 2 hours reaction at roomtemperature. The crude was dropwise added to 300 mL of ether forprecipitation and the solid was collected by centrifugation at 4400 rpmfor 10 minutes at room temperature. The crude product was resuspendedwith minimal volume of CH₂Cl₂ and dropwise added to 300 mL of ethercontaining 1.4 g of LiClO₄. The precipitate was collected bycentrifugation at 4400 rpm for 10 minutes at room temperature to affordthe white solid compound 11c; yield: 1.08 g (97.5%); ³¹P NMR (162 MHz,D₂O) δ 7.59; HRMS (ESI-TOF) calcd. for C₃₅H₃₀N₇OPNa [M+Na]⁺ 730.1791;found 730.1799.

3′-O, N⁶,N⁶-Tribenzoyl-2′-deoxyadenosine-5′-γ-(2-(pyrenesulfonyl)ethyl]-triphosphate(Compound 12c)

General procedure D with 1.08 g (1.34 mmol) of compound 11c, 0.77 g(1.63 mmol) of 1-(2-(pyrenesulfonyl)ethyl)-pyrophosphate (compound 7)and 13.6 mL (13.6 mmol) of ZnCl₂ solution (1.0 M in anhydrous DMF) for 3hours with stirring. After the reaction, the solution was precipitatedby 300 mL of ether. After centrifugation, the pellet was resuspended by20% H₂O/MeCN containing 2% Hunig's base and filtered by pyrex glassfunnel. The filtrate was evaporated and the crude product was purifiedby silica column chromatography with eluents [(8% H₂O/isopropanol+1%diisopropylethylamine), then (H₂O/(isopropanol-MeCN 1:1) from 2% to 7%containing 1% diisopropylethylamine (DIPEA)] to afford the white solidcompound 12c; yield: 928 mg (46.7%); TLC (H₂O/acetone 1:10 containing 2%DIPEA): Rf=0.28; ′H NMR (600 MHz, D₂O/DMSO-d₆ 1:1) δ 8.58-8.57 (m, 1H),8.45 (s, 1H), 8.37-8.33 (m, 2H), 8.06-7.70 (m, 11H), 7.57-7.32 (m, 7H),5.94 (t, 1H, J=7.2 Hz), 5.31 (s, 1H), 4.20 (d, 2H, J=6.6 Hz), 3.99-3.97(m, 2H), 3.86-3.84 (m, 1H), 3.75-3.74 (m, 1H), 2.55 (s, 1H), 2.10 (dd,1H, J=13.8, 4.8 Hz); ³¹P NMR (162 MHz, D₂O/DMSO-d₆ 1:1) δ −10.24 (d,J=14.9 Hz), −10.45 (d, J=14.7 Hz), −20.67 (brs, 1P); HRMS (ESI-TOF)calcd. for C₄₂H₃₄N₅O₁₆P₃SNa [M−2H+Na]⁺ 1012.0832; found 1012.0827.During HRMS, analysis one of the two benzoyl group is removed from theexocyclic amino group.

2′-deoxyadenosine-5′-triphosphate (compound βc)

General procedure E with 928 mg (0.63 mmol) of compound 12c in 45 mL of33% NH₄OH_((aq)) in deprotection step for 3 hours at 37° C. and then 15hours at room temperature. The product compound βc was afforded as awhite solid; yield: 284 mg (92.6%, A, ε₂₅₉: 15200 M⁻¹ cm⁻¹); ¹H NMR (400MHz, D₂O) δ 8.37 (s, 1H), 8.08 (s, 1H), 6.39 (t, 1H, J=6.4 Hz), 4.33 (s,1H), 4.26 (s, 2H), 2.81-2.74 (m, 1H), 2.66-2.60 (m, 1H); ³¹P NMR (162MHz, D₂O) δ −3.97 (d, J=15.7 Hz), −9.22 (d, J=16.0 Hz), −17.85 (t,J=13.3 Hz).

Synthesis of 2′-deoxyguanosine-5′-triphosphate

FIG. 68 shows the synthesis scheme for 2′-deoxyguanosine-5′-triphosphate(dGTP, compound βd). FIG. 69 through and FIG. 79 provide NMR spectra ofthe synthesized intermediates and product.

Methods used for the synthesis are now described.

3′-O, N²-Dibenzoyl-2′-deoxyguanosine-5′-dibenzylmonophosphate (Compound9d)

General procedure A with 1.0 g (2.10 mmol) of 3′-O,N²-dibenzoyl-2′-deoxyguanosine. 0.29 g (4.21 mmol) of tetrazole, 15 mLof anhydrous solution (MeCN/CH₂Cl₂, 1:1), 0.85 mL (2.52 mmol) ofdibenzyl-N, N-diisopropylphosphoramidite for 1 hour stirring at roomtemperature. Then, the reaction was added to 6 mL of H₂O₂ for 1 houroxidation reaction. Column chromatography with eluents (EtOAc/hexane,from 33% to 66%) to afford the product compound 9d as a white foam;yield: 1.35 g (85.9%); TLC (EtOAc/hexane, 1:60) R_(f)=0.32; ¹H NMR (400MHz, CDCl₃) δ 12.51 (s, 1H), 11.03 (s, 1H), 8.14 (d, 2H, J=6.0 Hz), 8.06(d, 2H, J=16.0 Hz), 7.73 (s, 1H), 7.62-7.60 (m, 2H), 7.50-7.45 (m, 4H),7.33 (m, 5H), 7.26-7.22 (m, 1H), 7.19-7.16 (m, 1H), 7.00 (d, 2H, J=16.0Hz), 6.25 (dd, 1H, J 12.0, 2.8 Hz), 5.70 (d, 1H, J 4.4 Hz), 5.08 (d, 2H,J 4.4 Hz), 4.80 (m, 2H), 4.56 (m, 1H), 4.43 (s, 1H), 4.35 (m, 1H), 3.60(m, 1H), 2.51 (s, 1H); ¹³C NMR (125.8 MHz, CDCl₃) δ 169.2, 166.0, 155.7,148.2, 148.1, 139.5, 135.4, 135.1, 135.0, 133.8, 133.2, 132.1, 129.8,129.3, 128.9, 128.8, 128.7, 128.6, 128.5, 128.3, 128.2, 128.2, 127.3,127.3, 123.5, 86.8, 83.2, 75.8, 69.9 (d, J C,P=5.3 Hz), 69.4 (d, JC,P=5.2 Hz), 66.8, 35.5; ³¹P NMR (162 MHz, CDCl₃) δ −0.55; HRMS(ESI-TOF) calcd. for C₃₈H₃₄N₅O₉PNa [M+Na]⁺ 758.1992; found 758.2000.

3′-O, N²-Dibenzoyl-2′-deoxyguanosine-5′-monophosphate (compound 10d)

General procedure B with 0.88 g (1.19 mmol) of compound 9d, 10 mL ofMeOH, and 250 mg of 10% Pd/C for 3 hours with stirring. The suspensionwas filtered over a pad of celite, washed with 100 mL of MeOH containing2% triethylamine four times to afford the product compound 10d as awhite foam of triethylammonium salt; yield: 0.72 g (84.5%); TLC(MeOH/CH₂Cl₂ 1:10 with 1% triethylamine); ¹H NMR (400 MHz, DMSO-d₆) δ8.28 (s, 1H), 8.18 (d, 2H, J=6.4 Hz), 8.03 (m, 2H), 7.70-7.62 (m, 1H),7.61-7.59 (m, 1H), 7.57-7.54 (m, 2H), 7.51 (m, 2H), 6.44 (m, 1H), 5.71(d, 1H, J=3.6 Hz), 4.38 (s, 1H), 4.12 (m, 1H), 4.02 (m, 1H), 3.35 (m,1H), 2.56 (dd, 1H, J=15.6, 6.8 Hz). ¹³C NMR (125.8 MHz, DMSO-d₆) δ169.8, 165.1, 155.2, 148.8, 139.2, 133.6, 132.8, 132.7, 129.4, 129.3,128.8, 128.7, 128.2, 128.0, 126.8, 121.1, 84.6, 83.7, 76.8, 63.9, 30.5;³¹P NMR (162 MHz, CD₃OD) δ −0.34; HRMS (ESI-TOF) calcd. forC₂₄H₂₁N₅O₉PNa₂ [M−H+2Na]⁺ 600.0872; found 600.0865.

3′-O, N²-Dibenzoyl-2′-deoxyguanosine-5′-phosphor-2-methylimidazolide(Compound 11d)

General procedure C with 0.65 g (1.17 mmol) of compound 10d, 10 mL ofanhydrous DMF, 0.82 mL (7.19 mmol) of triethylamine, 240 mg (5.85 mmol)of 2-methylimidazole, 768.3 mg (2.93 mmol) of triphenylphosphine, 645.5mg (2.93 mmol) of dipyridyl disulfide for 2 hours reaction at roomtemperature. First precipitation was achieved with 300 mL of diethylether. The product was resuspended with 15 mL of CH₂Cl₂ and dropwiseadded to the solution containing 1.17 g of sodium perchlorate, 15 mL oftriethylamine in 300 mL of ethyl acetate for second precipitation. Theproduct was afforded as a white solid compound 11d; yield: 0.68 g(93.4%); ³¹P NMR (162 MHz, D₂O) δ −8.70; HRMS (ESI-TOF) calcd. forC₂₈H₂₅N₇O₈PNa₂ [M−H+2Na]⁺ 664.1298; found 664.1276.

3′-O,N²-Dibenzoyl-2′-deoxyadenosine-5′-(γ-(2-(pyrenesulfonyl)ethyl))-triphosphate(Compound 12d)

General procedure D with 0.5 g (0.81 mmol) of compound 11d, 0.495 g(1.05 mmol) of 1-(2-(pyrenesulfonyl)ethyl)-pyrophosphate (compound 7)and 5.38 mL (8.07 mmol) of ZnCl₂ solution (1.5 M in anhydrous DMF) for 3h with stirring. After the reaction, the solution was precipitated by300 mL of ether. Silica column chromatography with eluents [(H₂O inisopropanol/MeCN) 1:1 from 3% to 8% containing 1% diisopropylethylamine(DIPEA)] to afford the white solid compound 12d; yield: 1.07 g (58.1%);TLC (H₂O/acetonitrile 1:10 containing 2% DIPEA) Rf=0.34; ′H NMR (400MHz, DMSO-d₆) δ 7.90-7.60 (m, 1H), 7.31 (s, 6H), 7.03-6.98 (m, 5H), 6.72(m, 3H), 6.52 (s, 4H), 5.32 (s, 1H), 4.62 (s, 1H), 3.36 (brs, 2H), 3.16(brs, 2H), 2.87 (s, 1H), 2.09 (s, 2H); ³¹P NMR (162 MHz, DMSO-d₆) δ−11.34, −12.12, −20.44; HRMS (ESI-TOF) calcd. for C₄₂H₃₄N₅O₁₇P₃SNa[M−2H+Na]1028.0781; found 1028.0796.

2′-deoxyguanosine-5′-triphosphate (Compound βd)

General procedure E with 800 mg (0.81 mmol) of compound 12d in 45 mL of33% NH₄OH(aq) for 3 hours at 37° C. and then 15 hours deprotectionreaction at room temperature. The product compound βd was afforded as awhite solid; yield: 372 mg (91.1%, G, ε₂₅₃: 13700 M⁻¹cm⁻¹); ¹H NMR (400MHz, D₂O) δ 8.15 (m, 1H), 6.32 (s, 1H), 4.27 (s, 1H), 4.19 (m, 2H), 4.15(s, 1H), 2.79 (brs, 1H), 2.52 (s, 1H); ³¹P NMR (162 MHz, D₂O) (−5.80 (d,J=15.7 Hz), −10.86 (d, J=16.0 Hz), −20.59 (t, J=13.3 Hz).

Synthesis of 2′-deoxythymidine-3′-triphosphate

FIG. 80 shows the synthesis scheme for 2′-deoxythymidine-3′-triphosphate(3′-TTP, compound 19). FIG. 81 through FIG. 91 provide NMR spectra ofthe synthesized intermediates and product.

Methods used for the synthesis are now described.

5′-Benzoyl-2′-deoxythymidine-3′-dibenzylmonophosphate (compound 15)

General procedure A with 1.04 g (3.0 mmol) of5′-benzoyl-2′-deoxythymidine compound 14, 383 mg (5.4 mmol) oftetrazole, 30 mL of anhydrous solution (MeCN/CH₂Cl₂, 1:1), 1.23 mL (3.91mmol) of dibenzyl-N, N-diisopropylphosphoramidite for 3 hours reactionat room temperature. Then 8 mL of 30% H₂O_(2(aq)) for 1 hour reaction atroom temperature. Column chromatography with eluents (MeOH/CH₂Cl₂, from1% to 1.4%) to afford the product compound 15 as a white solid; yield:1.28 g (70.3%); TLC (MeOH/CH₂Cl₂, 1:40) R_(f)=0.22; ¹H NMR (400 MHz,CDCl₃) δ 9.46 (s, 1H), 7.97 (d, 2H, J=8.0 Hz), 7.61-7.57 (m, 1H), 7.45(t, 2H, J=7.2 Hz), 7.35-7.31 (m, 10H), 7.08 (s, 1H), 6.25 (dd, 1H,J=7.6, 6.4 Hz), 5.11-4.97 (m, 5H), 4.56 (dd, 1H, J=13.2, 3.6 Hz),4.33-4.29 (m, 2H), 2.48 (dd, 1H, J=14.4, 5.6 Hz), 2.11-2.06 (m, 1H),1.57 (s, 3H); ¹³C NMR (125.8 MHz, CDCl₃) δ 165.7, 163.8, 150.4, 135.3,135.2, 134.3, 133.5, 129.3, 129.2, 128.7, 128.7, 128.6, 128.6, 128.1,128.1, 111.4, 84.5, 82.6 (d, J C,P=5.4 Hz), 69.8, 69.8, 69.8, 63.6, 38.6(d, J C,P=4.7 Hz), 12.0; ³¹P NMR (162 MHz, CDCl₃) δ −0.31; HRMS(ESI-TOF) calcd. for C₃₁H₃₁N₂O₉PNa [M+Na]⁺ 629.1665; found 629.1666.

5′-Benzoyl-2′-deoxythymidine-3′-monophosphate (compound 16)

General procedure B with 1.28 g (2.11 mmol) of compound 15, 50 mL ofMeOH, and 200 mg of 10% Pd/C for 3 hours with stirring at roomtemperature. The suspension was filtered over a pad of celite, washedwith 100 mL of MeOH four times to afford the product, compound 16, as awhite foam; yield: 0.86 g (96.2%); TLC (MeOH/CH₂Cl₂, 1:10) R_(f)=0; ¹HNMR (400 MHz, CD₃OD) δ 8.06 (d, 2H, J=7.2 Hz), 7.64 (t, 1H, J=7.6 Hz),7.51 (t, 2H, J=7.6 Hz), 7.39 (s, 1H), 6.29 (m, 1H), 5.10 (t, 1H, J=6.8Hz), 4.70 (dd, 1H, J=12.0, 3.2 Hz), 4.57-4.47 (m, 2H), 2.63 (ddd, 1H,J=14.0, 6.0, 2.4 Hz), 2.43 (m, 1H), 1.62 (s, 3H); ¹³C NMR (125.8 MHz,CD₃OD) δ 167.4, 166.1, 152.1, 137.0, 134.6, 130.8, 130.5, 129.8, 111.8,86.4, 84.6, 77.2, 65.1, 39.5, 12.2; ³¹P NMR (162 MHz, CD₃OD) δ 0.42;HRMS (ESI-TOF) calcd. for C₁₇H₁₈N₂O₉PNa₂ [M−H+2Na]⁺ 471.0545; found471.0551.

5′-Benzoyl-2′-deoxythymidine-3′-phosphor-2-methylimidazolide (Compound17)

General procedure C with 0.86 g (2.0 mmol) of compound 16, 10 mL ofanhydrous DMF, 1.39 mL (10.0 mmol) of triethylamine, 328 mg (4.1 mmol)of 2-methylimidazole, 1.05 g (4.0 mmol) of triphenylphosphine, 903 g(4.0 mmol) of dipyridyl disulfide. First precipitation was achieved in250 mL of diethyl ether. The product was resuspended with 10 mL of DMFand dropwise added to the solution containing 2.0 g of sodiumperchlorate, 20 mL of triethylamine in 400 mL of ethyl acetate for thesecond precipitation. The product was afforded as a white solid compound17; yield: 0.89 g (86.9%); ³¹P NMR (162 MHz, D₂O) δ −7.57; HRMS(ESI-TOF) calcd. for C₂₁H₂₃N₄O₈PNa [M+Na]⁺ 513.1151; found 513.1158.

5′-Benzoyl-2′-deoxythymidine-3′-γ-(2-(pyrenesulfonyl)ethyl)triphosphate(Compound 18)

General procedure D with 0.89 g (1.74 mmol) of compound 16, 0.89 g (1.90mmol) of 1-(2-(pyrenesulfonyl)ethyl)) pyrophosphate (compound 7) and11.5 mL (17.30 mmol) of ZnCl₂ solution (1.5 M in anhydrous DMF) for 3hours with stirring at room temperature. The product was purified by 2.5cm silica gel flash column with eluents [H₂O-isopropanol from 5% to 10%containing 1% diisopropylethylamine (DIPEA)] to afford the yellowishsolid compound 18; yield: 1.23 g (55.8%); TLC (1:10H₂O-acetone/2%-DIPEA): Rf=30.8; ′H NMR (400 MHz, D₂O) δ 8.25 (d, 1H,J=8.8 Hz), 8.11 (d, 1H, J=6.4 Hz), 7.47 (d, 1H, J=8.0 Hz), 7.36-7.34 (m,4H), 7.15-7.07 (m, 2H), 6.94-6.79 (m, 5H), 6.26 (s, 1H), 5.12 (s, 1H),4.85 (s, 1H), 4.48-4.25 (m, 5H), 3.80 (s, 2H), 2.35 (s, 1H), 1.84 (s,1H), 0.66 (s, 3H); ³¹P NMR (162 MHz, D₂O) (−10.56 (d, 1P, J=12.5 Hz),−11.07 (d, 1P, J=14.1 Hz), −20.85 (brs, 1P); HRMS (ESI-TOF) calcd. forC₃₅H₃₀N₂O₁₇P₃SNa₄ [M−3H+4Na]⁺ 967.0069; found 967.0080.

2′-deoxythymidine-3′-triphosphate (Compound 19)

General procedure E with 1.23 g of compound 18, 5 mL of CH₂Cl₂ and 50 mLof 33% NH₄OH_((aq)) for 18 hours deprotection at room temperature. Theproduct, compound 19, was afforded as a white solid; yield: 404 mg (86%,ε₂₆₇=9600 M⁻¹cm⁻¹); ¹H NMR (400 MHz, D₂O) δ 7.74 (s, 1H), 6.40 (t, 1H,J=7.6 Hz), 5.05 (s, 1H), 4.34 (d, 1H, J=2.8 Hz), 3.92 (d, 2H, J=3.6 Hz),2.70-2.65 (m, 1H), 2.54-2.47 (m, 1H), 1.96 (s, 3H); ³¹P NMR (162 MHz,D₂O) (−4.03 (d, 1P, J=17.0 Hz), −9.99 (d, 1P, J=16.7 Hz), −18.47 (brs,1P).

Synthesis of 1-(α-L-threofuranosyl)thymidine-3′-triphosphate

FIG. 92 shows the synthesis scheme for1-(α-L-threofuranosyl)thymidine-3′-triphosphate (tTTP, compound 25a).FIG. 93 through FIG. 100 provide NMR spectra of the synthesizedintermediates and product.

Methods used for the synthesis are now described.

1-(2′-O-benzoyl-α-L-threofuranosyl)thymidine-3′-dibenzylmonophosphate(compound 21a)

General procedure A with 1 g (3.01 mmol) of1-(2′-O-benzoyl-α-L-threofuranosyl)thymine, 379.7 mg (5.42 mmol) oftetrazole, 24 mL of anhydrous solution (MeCN/CH₂Cl₂, 1:1), 1.24 mL (3.91mmol) of dibenzyl-N, N-diisopropylphosphoramidite for 3 hours stirringat room temperature and 6 mL of H₂O₂ for 1 hour oxidation reaction.Silica gel column chromatography with eluents (MeOH/CH₂Cl₂, from 0 to2%). The product, compound 21a, was acquired as a white solid; yield:1.25 g (70.1%). Compound 21a has been reported in Sau et al., 2017, Org.Lett. 19, 4379-4382.

1-(2′-O-benzoyl-α-L-threofuranosyl)thymidine-3′-monophosphate (compound22a)

General procedure B with 1.25 g (2.11 mmol) of compound 21a, 50 mL ofMeOH, and 200 mg of 10% Pd/C for 3 hours stirring. The suspension wasfiltered over a pad of celite, washed with 100 mL of MeOH four times.The product compound 22a was afforded as a white foam; yield: 0.83 g(92.4%); TLC (MeOH/CH₂Cl₂, 1:10) R_(f)=0; ¹H NMR (400 MHz, CD₃OD) δ 8.05(d, 1H, J=7.6 Hz), 7.64 (t, 1H, J=7.2 Hz), 7.57 (s, 1H), 7.50 (t, 2H,J=7.2 Hz), 6.05 (s, 1H), 5.60 (s, 1H), 5.01 (s, 1H), 4.52 (d, 1H, J=10.4Hz), 4.29 (d, 1H, J=9.2 Hz), 1.92 (s, 3H); ¹³C NMR (125.8 MHz, CD₃OD) δ166.3, 152.2, 138.0, 134.9, 130.9, 130.1, 129.7, 111.7, 91.2, 82.1 (d, JC,P=5.9 Hz), 79.0, 74.7, 12.5; ³¹P NMR (162 MHz, CDCl₃) δ −0.23; HRMS(ESI-TOF) calcd. for C₁₆H₁₇N₂O₉PNa [M+Na]⁺ 435.0569; found 435.0557.

1-(2′-O-benzoyl-α-L-threofuranosyl)thymidine-3′-monophosphor-2-methylimidazolide(Compound 23a)

General procedure C with 0.83 g (2 mmol) of compound 22a, 7.0 mL ofanhydrous DMF, 1.40 mL (10.1 mmol) of triethylamine, 328 mg (4 mmol) of2-methylimidazole, 1.05 g (4 mmol) of triphenylphosphine, 0.88 g (4mmol) of dipyridyl disulfide. First precipitation was achieved with 250mL of diethyl ether. The product was resuspended with 15 mL of CH₂Cl₂and dropwise added to the solution containing 2.01 g of sodiumperchlorate, 10 mL of triethylamine in 300 mL of ethyl acetate for thesecond precipitation. The product, compound 23a, was afforded as a whitesolid; yield: 0.98 g (98.3%); ³¹P NMR (162 MHz, D₂O) δ −8.17; HRMS(ESI-TOF) calcd. for C₂₀H₂₁N₄O₈PNa [M+Na]⁺ 499.0995; found 499.0992.

1-(2′-O-benzoyl-α-L-threofuranosyl)thymidine-3′-(γ-(2-(pyrenesulfonyl)ethyl))triphosphate (Compound 24a)

General procedure D with 0.98 g (1.97 mmol) of compound 23a, 1.11 g(2.36 mmol) of compound 7 and 13.13 mL (19.7 mmol, 1.5 M in anhydrousDMF) of ZnCl₂ solution for 3 hours stirring at room temperature. Theproduct was purified by 2 cm silica gel column chromatography witheluents [H₂O-isopropanol from 5% to 12.5% containing 1%diisopropylethylamine (DIPEA)] to afford the white solid compound 24a;yield: 1.53 g (62.1%); TLC (1:10 H₂O-acetone with 2%-DIPEA): Rf=0.25; ′HNMR (400 MHz, D₂O) δ 8.22 (d, 1H, J=9.2 Hz), 8.09 (d, 1H, J=8.0 Hz),7.48-7.30 (m, 7H), 7.09-6.98 (m, 4H), 6.84 (t, 2H, J=6.8 Hz), 5.32 (s,1H), 5.20 (s, 1H), 4.89 (s, 1H), 4.48-4.40 (m, 3H), 3.97 (d, 1H, J=8.0Hz), 3.76 (m, 2H), 1.50 (s, 3H); ³¹P NMR (162 MHz, D₂O) δ −10.23 (d,J=13.0 Hz), −11.65 (d, J=13.9 Hz), −20.59 (brs); HRMS (ESI-TOF) calcd.for C₃₄H₂₉N₂O₁₇P₃SNa₃ [M−2H+3Na]⁺ 931.0093; found 931.0049.

1-(α-L-threofuranosyl)thymidine-3′-triphosphate (compound 25a)

General procedure E with 1.53 g (1.22 mmol) of compound 24a in 50 mL of33% NH₄OH_((aq)) for 18 hours deprotection at room temperature. Theproduct, compound 25a, was afforded as a white solid; yield: 445 mg(78.9%, ε₂₆₇=9600 M⁻¹cm⁻¹); ¹H NMR (400 MHz, D₂O) δ 7.62 (s, 1H), 5.85(s, 1H), 4.90 (s, 1H), 4.61 (s, 1H), 4.51 (d, 1H, J=10.4 Hz), 4.40 (d,1H, J=9.2 Hz), 1.95 (s, 1H); ³¹P NMR (162 MHz, D₂O) δ −4.89 (d, J=14.7Hz), −11.02 (d, J=18.63 Hz), −19.80 (brs).

Synthesis of 1-(α-L-threofuranosyl)cytidine-3′-triphosphate

FIG. 101 shows the synthesis scheme for1-(α-L-threofuranosyl)cytidine-3′-triphosphate (tCTP, compound 25b).FIG. 102 through FIG. 109 provide NMR spectra of the synthesizedintermediates and product.

Methods used for the synthesis are now described.

N⁴-Benzoyl-1-(2′-O-benzoyl-α-L-threofuranosyl)cytidine-3′-dibenzylmonophosphate(Compound 21b)

Modified general procedure A with 1 g (2.37 mmol) ofN⁴-benzoyl-1-(2′-O-benzoyl-α-L-threofuranosyl)cytosine (compound 20b),300 mg (4.27 mmol) of tetrazole, 15.8 mL of anhydrous solution(MeCN/CH₂Cl₂, 1:1), 0.98 mL (3.09 mmol) of dibenzyl-N,N-diisopropylphosphoramidite for 1 hour reaction at room temperature and5 mL of H₂O₂ for 1 hour oxidation reaction. After the reaction, theproduct, compound 21b, was afforded as a white solid; yield: 1.29 g(79.3%).

N⁴-Benzoyl-1-(2′-O-benzoyl-α-L-threofuranosyl)cytidine-3′-monophosphate(Compound 22b)

General procedure B with 1.29 g (1.89 mmol) of compound 21b, 50 mL ofMeOH, and 250 mg of 10% Pd/C for 5 hours stirring. The suspension wasfiltered over a pad of celite, washed with 100 mL of MeOH containing 2%triethylamine four times. The product, compound 22b, was afforded as awhite foam of a triethylammonium salt; yield 1.23 g (92.8%); ¹H NMR (400MHz, CDCl₃) δ 8.26 (d, 1H, J=7.6 Hz), 7.99 (d, 2H, J=7.2 Hz), 7.90 (d,2H, J=7.6 Hz), 7.57-7.53 (m, 2H), 7.47-7.39 (m, 5H), 6.18 (s, 1H), 5.66(s, 1H), 4.91-4.85 (m, 2H), 4.28 (dd, 1H, J=10.4, 3.2 Hz); ¹³C NMR(125.8 MHz, CDCl₃) δ 164.6, 162.7, 154.9, 146.2, 133.5, 132.9, 129.9,129.3, 128.9, 128.9, 128.5, 128.0, 128.0, 96.6, 91.4, 80.9 (d, J C,P=6.5Hz), 76.4 (d, J C,P=4.7 Hz), 57.9; ³¹P NMR (162 MHz, CDCl₃) δ −1.15;HRMS (ESI-TOF) calcd. for C₂₀H₂ON₃O₉PNa₃ [M−2H+3Na]⁺ 546.0630; found546.0640.

N⁴-Benzoyl-1-(2′-O-benzoyl-α-L-threofuranosyl)cytidine-3′-monophosphor-2-methylimidazolide(Compound 23b)

General procedure C with 1.02 g (1.75 mmol) of compound 22b, 4.3 mL ofanhydrous DMF, 0.87 mL (6.4 mmol) of triethylamine, 287 mg (3.5 mmol) of2-methylimidazole, 0.94 g (3.6 mmol) of triphenylphosphine, 0.79 g (3.6mmol) of dipyridyl disulfide for 2 hours stirring at room temperature.First precipitation was achieved with 200 mL of diethyl ether. Theproduct was resuspended with 15 mL of CH₂Cl₂ and dropwise added to thesolution containing 2.04 g of sodium perchlorate, 10 mL of triethylaminein 300 mL of ethyl acetate for the second precipitation. The product,compound 23b, was afforded as a white solid; yield: 0.94 g (91.5%); ³¹PNMR (162 MHz, D₂O) δ −7.56; HRMS (ESI-TOF) calcd. for C₂₆H₂₄N₅O₈PNa[M+Na]⁺ 588.1260; found 588.1246.

N⁴-Benzoyl-1-(2′-O-benzoyl-α-L-threofuranosyl)cytidine-3′-(γ-(2-(pyrenesulfonyl)ethyl)triphosphate (Compound 24b)

General procedure D with 0.94 g (1.6 mmol) of compound 23b, 0.9 g (1.92mmol) of compound 7 and 10.67 mL (16 mmol, 1.5 M in anhydrous DMF) ofZnCl₂ solution for 5 hours stirring at room temperature. The product waspurified by silica gel chromatography with eluents [(6% H₂O/isopropanolcontaining 1% DIPEA) and then (5% to 10% of H₂O/(isopropanol-MeCN 1:1))containing 1% diisopropylethylamine (DIPEA) to afford the white solidcompound 24b; yield: 1.43 g (66.7%); TLC (1:10 H₂O-acetone with2%-DIPEA) Rf=0.25; ′H NMR (400 MHz, D₂O) δ 8.18 (s, 1H), 8.04 (s, 1H),7.63-7.25 (m, βH), 7.14 (s, 3H), 6.95-6.91 (m, 4H), 6.64 (s, 1H), 5.57(s, 1H), 5.41 (s, 1H), 4.94 (s, 1H), 4.63 (s, 1H), 4.44 (s, 2H), 4.23(s, 1H), 3.74-3.63 (m, 2H); ³¹P NMR (162 MHz, D₂O) δ −10.23 (d, J=13.0Hz), −11.65 (d, J=13.9 Hz), −20.59 (brs); HRMS (ESI-TOF) calcd. forC₄₀H₃₂N₃O₁₇P₃SNa [M+Na]⁺ 974.0563; found 974.0620.

1-(α-L-threofuranosyl)cytidine-3′-triphosphate (compound 25b)

General procedure E with 1.43 g (1.07 mmol) of compound 24b in 50 mL of33% NH₄OH_((aq)) for 3 hours at 37° C. and then 15 hours deprotectionreaction at room temperature. The product, compound 25b, was afforded asa white solid; yield: 412 mg (85.8%, 6280=13100 M⁻¹cm⁻¹); ¹H NMR (400MHz, D₂O) δ 7.82 (d, 1H, J=7.6 Hz), 6.09 (d, 1H, J=7.6 Hz), 5.90 (s,1H), 4.91 (d, 1H, J=4.8 Hz), 4.58-4.53 (m, 2H), 4.42 (d, 1H, J=6.8 Hz);³¹P NMR (162 MHz, D₂O) δ −4.26 (brs), −10.93 (d, J=17.3 Hz), −19.15(brs).

Synthesis of 9-(α-L-threofuranosyl)adenosine-3′-triphosphate

FIG. 110 shows the synthesis scheme for9-(α-L-threofuranosyl)adenosine-3′-triphosphate (tATP, compound 25c).FIG. 111 through FIG. 118 provide NMR spectra of the synthesizedintermediates and product.

Methods used for the synthesis are now described.

N⁶-Benzoyl-9-(2′-O-benzoyl-α-L-threofuranosyl)adenosine-3′-dibenzylmonophosphate (Compound 21c)

General procedure A with 1.007 g (2.26 mmol) ofN⁶-benzoyl-9-(2′-O-benzoyl-α-L-threofuranosyl)adenine (compound 20c),316 mg (4.52 mmol) of tetrazole, 15 mL of anhydrous solution(MeCN/CH₂Cl₂, 1:1), 0.91 mL (2.71 mmol) of dibenzyl-N,N-diisopropylphosphoramidite for 3 hours reaction at room temperatureand 5 mL of H₂O₂ for 1 hour oxidation reaction. The product, compound21c, was afford as a white solid; yield: 1.41 g (88.4%).

N⁶-Benzoyl-9-(2′-O-benzoyl-α-L-threofuranosyl)adenosine-3′-monophosphate(compound 22c)

General procedure B with 1.3 g (1.84 mmol) of compound 21c, 10 mL ofMeOH, and 400 mg of 10% Pd/C for 12 hours with stirring. The suspensionwas filtered over a pad of celite, washed with 100 mL of MeOH fourtimes. The product, compound 22c, was afford as a white solid; yield:943 mg (97.4%); ¹H NMR (400 MHz, CD₃OD): δ 8.98 (s, 1H), 8.91 (s, 1H),8.26-8.17 (m, 4H), 7.83-7.80 (m, 2H), 7.74-7.65 (m, 4H), 6.68 (s, 1H),6.18 (s, 1H), 5.33 (s, 1H), 4.88-4.85 (m, 1H), 4.66 (dd, 1H, J=14, 6.4Hz). ¹³C NMR (125.8 MHz, DMSO-d₆): δ 164.7, 151.9, 150.4, 142.8, 133.9,133.3, 132.4, 129.6, 129.0, 128.7, 128.4, 128.4, 125.3, 109.4, 87.0,80.3, 76.2, 72.6; ³¹P NMR (162 MHz, DMSO-d₆): 0.30; HRMS (ESI-TOF)calcd. for C₂₃H₂₁N₅O₈P [M+H]⁺ 526.1128; found 526.1118.

N⁶—Benzoyl-9-(2′-O-benzoyl-α-L-threofuranosyl)adenosine-3′-monophosphor-2-methylimidazolide(compound 23c)

General procedure C with 0.93 g (1.58 mmol) of compound 22c, 12 mL ofanhydrous DMF, 1.1 mL (7.89 mmol) of triethylamine, 284 mg (3.94 mmol)of 2-methylimidazole, 1.03 g (3.94 mmol) of triphenylphosphine, 0.87 g(3.95 mmol) of dipyridyl disulfide. First precipitation was achievedwith 150 mL of diethyl ether. The product was resuspended with 5 mL ofCH₂Cl₂ and dropwise added to the solution containing 1.5 g of sodiumperchlorate, 6 mL of triethylamine in 150 mL of ethyl acetate for thesecond precipitation. The product afford as a white solid compound 23c;yield: 0.89 g (93.02%); ³¹P NMR (162 MHz, DMSO-d₆): −9.80; HRMS(ESI-TOF) calcd. for C₂₇H₂₄N₇O₇PNa [M+Na]⁺ 612.1373; found 612.1345.

N⁶-Benzoyl-9-(2′-O-benzoyl-α-L-threofuranosyl)adenosine-3′-γ-[2-(pyrenesulphonyl)ethyl]triphosphate (Compound 24c)

General procedure D with 0.6 g (1.02 mmol) of compound 23c, 0.52 g(mmol) of compound 7 and 7 mL (10.17 mmol, 1.5 M in anhydrous DMF) ofZnCl₂ solution for 3 hours stirring at room temperature. The product waspurified by silica gel chromatography with eluents [MeOH—CHCl₃ from 5%to 12% containing 1% diisopropylethylamine (DIPEA)] to afford the whitesolid compound 24c; yield 0.64 g (63.1%); TLC (1:10 H₂O-acetone with2%-DIPEA) Rf=0.31; ′H NMR (400 MHz, D₂O) δ 9.50 (s, 1H), 9.18-7.56 (m,7H), 7.51-7.46 (m, 6H), 7.35-7.09 (m, 9H), 6.19 (m, 1H), 5.72 (m, 1H),4.72-4.52 (m, 1H), 3.48 (s, 2H), 2.99 (d, 2H, J=12 Hz); ³¹p NMR (162MHz, D₂O) δ −9.21 (d, J=12.9 Hz), −10.51 (d, J=13.0 Hz), −19.24 (brs);HRMS (ESI-TOF) calcd. for C₄₁H₃₂N₅O₁₆P₃SNa [M−2H+Na]⁻ 998.0676; found998.0667.

9-(α-L-threofuranosyl)adenosine-3′-triphosphate (Compound 25c)

General procedure E with 0.5 g (0.51 mmol) of compound 24c, 35 mL of 33%NH₄OH_((aq)) for 3 hours deprotection at 37° C., and then 15 hoursdeprotection reaction at room temperature. The product compound 25c wasafforded as a white solid; yield: 0.21 g (86.2%, ε₂₈₀=15200 M“cm”); ¹HNMR (400 MHz, D₂O) δ 8.36 (s, 1H), 8.27 (s, 1H), 6.16 (s, 1H), 5.09 (d,1H, J=8.0 Hz), 4.96 (s, 1H), 4.50 (s, 2H); ³¹P NMR (162 MHz, D₂O) δ−4.02 (d, J=16.6 Hz), −10.51 (d, J=16.2 Hz), −17.77 (brs).

Synthesis of 9-(α-L-threofuranosyl)guanosine-3′-triphosphate

FIG. 119 shows the synthesis scheme for9-(α-L-threofuranosyl)guanosine-3′-triphosphate (tGTP, compound 25d).FIG. 120 through FIG. 127 provide NMR spectra of the synthesizedintermediates and product.

Methods used for the synthesis are now described.

N²-Acetyl-9-(2′-O-benzoyl-α-L-threofuranosyl)guanosine-3′-dibenzylmonophosphate(Compound 21d)

General procedure A with 1 g (1.68 mmol) ofN²-acetyl-9-(2′-O-benzoyl-α-L-threofuranosyl)guanine (compound 20d), 212mg (3.03 mmol) of tetrazole, 12 mL of anhydrous solution (MeCN/CH₂Cl₂,1:1), 735 mL (2.19 mmol) of dibenzyl-N, N-diisopropylphosphoramidite for3 hours reaction at room temperature and 6 mL of H₂O₂ for 1 houroxidation. Column chromatography with eluents (EtOAc/Hexane, 50%; thenEtOAc/CH₂Cl₂, from 16% to 25%) to afford the product compound 21d as awhite solid; yield: 1.14 g (79.4%). TLC (EtOAc/CH₂Cl₂, 1:4) Rf=0.44;Compound 20d has been reported in Sau et al., 2016, J. Org. Chem. 81,2302-2307. Compound 21d has been reported in Sau et al., 2017, Org.Lett. 19, 4379-4382.

N²-Acetyl-9-(2′-O-benzoyl-α-L-threofuranosyl)guanosine-3′-monophosphate(Compound 22d)

General procedure B with 1.63 g (2.47 mmol) of compound 21d, 15 mL ofMeOH, and 600 mg of 10% Pd/C for 3 hours stirring. The suspension wasfiltered over a pad of celite, washed with 100 mL of MeOH four times.The product compound 22d was afford as a white solid; yield; 1.04 g(87.7%). ¹H NMR (400 MHz, DMSO-d₆): δ 12.1 (s, 1H), 11.68 (s, 1H), 8.13(s, 1H), 8.04-8.02 (m, 2H), 7.74-7.70 (m, 1H), 7.59-7.55 (m, 2H), 6.11(d, 1H, J=4 Hz), 5.86 (s, 1H), 5.09 (d, 1H, J=2.4 Hz) 4.47-4.45 (m, 1H),4.38-4.34 (m, 1H), 2.15 (s, 3H); ¹³C NMR (125.8 MHz, DMSO-d₆): 173.5,164.5, 154.8, 148.4, 148.1, 137.7, 134.1, 129.6, 128.9, 128.3, 119.9,86.9, 80.6, 76.5, 72.8, 48.6, 23.8; ³¹P NMR (162 MHz, DMSO-d₆): −0.89;HRMS (ESI-TOF) calcd. for C₁₈H₁SN₅O₉PNa [M+Na]⁺ 502.0740; found502.0727.

N²-Acetyl-9-(2′-O-benzoyl-α-L-threofuranosyl)guanosine-3′-monophosphor-2-methylimidazolide(Compound 23d)

General procedure C with 0.94 g (1.97 mmol) of compound 22d, 12 mL ofanhydrous DMF, 1.3 mL (9.86 mmol) of triethylamine, 355 mg (4.93 mmol)of 2-methylimidazole, 1.29 g (4.93 mmol) of triphenylphosphine, 1.086 g(4.93 mmol) of dipyridyl disulfide. First precipitation was achievedwith 150 mL of diethyl ether. The product was resuspended with 5 mL ofCH₂Cl₂ and dropwise added to the solution containing 1.5 g of sodiumperchlorate, 6 mL of trimethylamine in 150 mL of ethyl acetate for thesecond precipitation. The product afford as a white solid compound 23d;yield: 0.88 g (82.1%); ³¹P NMR (162 MHz, DMSO-d₆):-9.77; HRMS (ESI-TOF)calcd. for C₂₂H₂₂N₇O₈PNa [M+Na]⁺ 566.1165; found 566.1163.

N²-Acetyl-9-(2′-O-benzoyl-α-L-threofuranosyl)guanosine-3′-(γ-(2-(pyrenesulfonyl)ethyl))triphosphate (Compound 24d)

General procedure D with 0.5 g (0.92 mmol) of compound 23d, 0.56 g (1.20mmol) of 7 and 10 mL (9.2 mmol, 1.5 M in anhydrous DMF) of ZnCl₂solution for 3 hours stirring at room temperature. The product waspurified by silica gel chromatography with eluents [(H₂O/isopropanolfrom 5% to 10% then H₂O/(isopropanol-MeCN 1:1) containing 1%diisopropylethylamine (DIPEA)] to afford the white solid compound 24d;yield: 0.74 g (80.9%); TLC (1:10 H₂O-acetonitrile with 2% DIPEA)Rf=0.31; ′H NMR (400 MHz, D₂O) δ 12.00 (m, 1H), 8.96 (d, 1H, J=8.4 Hz),8.65 (d, 1H, J=8.2 Hz), 8.48-8.42 (m, 3H), 8.39-8.30 (m, 1H), 8.28 (s,1H), 8.19-8.11 (m, 2H), 7.99-7.97 (m, 1H), 7.51 (brs, 2H), 7.25 (s, 2H),7.13 (s, 2H), 7.01 (s, 1H), 6.01 (s, 1H), 5.85 (s, 1H), 5.09 (s, 1H),4.63 (s, 1H), 4.19 (s, 2H), 3.90 (d, 2H, J=5.6 Hz), 3.07 (s, 2H), 1.93(s, 3H); ³¹P NMR (162 MHz, D₂O) δ −12.87 (brs, 2P), −20.71 (brs, 1P);HRMS (ESI-TOF) calcd. for C₃₆H₃₀N₅O₁₇P₃SNa [M−2H+Na]⁻ 952.0473; found952.0466.

9-(α-L-threofuranosyl)guanosine-3′-triphosphate (Compound 25d)

General procedure E with 0.55 g of compound 24c, 50 mL of 33%NH₄OH_((aq)) for 3 hours deprotection at 37° C. and then 15 hours atroom temperature. The product was afforded as a white solid; yield: 0.24g (87.2%, ε₂₅₃=13700 M⁻¹cm⁻¹); ′H NMR (400 MHz, D₂O) δ 7.84 (m, 1H),6.06 (d, 1H, J=7.2 Hz), 5.84 (1H), 4.53 (d, 1H, J=8 Hz), 4.41 (m, 1H),4.31 (s, 2H), 3.35 (m, 2H), 2.96 (m, 1H); ³¹P NMR (162 MHz, D₂O) δ −9.75(d, J=20.4 Hz), −11.27 (d, J=20.4 Hz), −22.16 (d, J=16.2 Hz).

Synthesis of L-2′-deoxythymidine-5′-triphosphate

FIG. 128 shows the synthesis scheme forL-2′-deoxythymidine-5′-triphosphate (L-dTTP, compound 31). FIG. 129through FIG. 139 provide NMR spectra of the synthesized intermediatesand product.

Methods used for the synthesis are now described.

3′-Benzoyl-2′-deoxy-L-thymidine-5′-dibenzylmonophosphate (Compound 27)

General procedure A with 200 mg (0.58 mmol) of compound 26, 72.9 mg(1.18 mmol) of tetrazole, 12 mL of anhydrous solution (MeCN/CH₂Cl₂,1:1), 260 μL (0.75 mmol) of dibenzyl-N, N-diisopropylphosphoramidite for3 hours reaction at room temperature. Then 2 mL of 30% H₂O_(2(aq)) for 1hour oxidation reaction at room temperature. Column chromatography witheluents (MeOH/CH₂Cl₂, from 1% to 1.4%) to afford the product compound 27as a white solid; yield: 0.32 g (68.2%); TLC (MeOH/CH₂Cl₂, 1:40)R_(f)-0.23; ′H NMR (400 MHz, CD₃OD) δ 7.96 (d, 2H, J=7.6 Hz), 7.55-7.52(m, 1H), 7.41-7.38 (m, 3H), 7.31-7.25 (m, 10H), 6.30 (dd, 1H, J=14.4,2.4 Hz), 5.35 (d, 1H, J=6.8 Hz), 5.06-5.03 (m, 4H), 4.31-4.28 (m, 2H),4.24 (brs, 1H), 2.42 (dd, J=19.2, 8.4 Hz), 2.21-2.14 (m, 1H), 1.72 (s,3H); βCNMR (125.8 MHz, CD₃OD) δ 167.9, 166.7, 152.9, 137.8, 137.7,137.7, 137.7, 137.6, 135.4, 131.5, 131.3, 130.6, 130.5, 130.5, 130.0,130.0, 112.9, 87.0, 84.7 (d, J C,P=6.5 Hz), 76.9, 71.8 (t, J C,P=4.5Hz), 69.4 (d, J C,P=4.6 Hz), 38.6, 13.4 (d, J C,P=2.7 Hz); ³¹P NMR (162MHz, CD₃OD) δ 0.19; HRMS (ESI-TOF) calcd. for C₃₁H₃₁N₂O₉PNa [M+Na]⁺629.1665; found 629.1685.

3′-Benzoyl-2′-deoxy-L-thymidine-5′-monophosphate (compound 28)

General procedure B with 300 mg (0.49 mmol) of compound 27, 15 mL ofMeOH, and 80 mg of 10% Pd/C for 3 hours stirring at room temperature.The suspension was filtered over a pad of celite, and washed with 60 mLof MeOH containing 2% triethylamine four times to afford the productcompound 28 as a white foam of triethylammonium salt; yield: 190 mg(91.3%); TLC (MeOH/CH₂Cl₂, 1:10 with 1% triethylamine) R_(f)=0; ¹H NMR(400 MHz, CD₃OD) δ 8.05-7.89 (m, 3H), 7.64-7.50 (m, 3H), 6.47 (s, 1H),5.66 (s, 1H), 4.40 (brs, 2H), 3.33-3.26 (m, 2H), 2.53 (s, 2H), 1.96 (s,3H); ¹³C NMR (125.8 MHz, CD₃OD) δ 168.1, 167.3, 153.4, 138.9, 135.3,131.8, 131.4, 130.5, 113.2, 86.9, 86.3, 78.8, 67.0, 48.2, 39.3, 13.4;³¹P NMR (162 MHz, CD₃OD) δ 1.02; HRMS (ESI-TOF) calcd. for C₁₀H₁₄N₂O₈P[M−H]-321.0488; found 321.0493.

3′-Benzoyl-2′-deoxy-L-thymidine-5′-phosphor-2-methylimidazolide(Compound 29)

General procedure C with 180 mg (0.42 mmol) of compound 28, 8 mL ofanhydrous DMF, 300 μL (2.1 mmol) of triethylamine, 86 mg (1.05 mmol) of2-methylimidazole, 275 mg (1.05 mmol) of triphenylphosphine, 242 mg(1.05 mmol) of dipyridyl disulfide for 2 hours reaction at roomtemperature. First precipitation was achieved with 50 mL of diethylether. The product was resuspended with 10 mL of DMF and dropwise addedto the solution containing 700 mg of sodium perchlorate, 15 mL oftriethylamine in 100 mL of ethyl acetate for the second precipitation.The product was afforded as a white solid compound 29; yield: 145 mg(96.9%); ³¹P NMR (162 MHz, CD₃OD) δ −7.85; HRMS (ESI-TOF) C₂₁H₂₃N₄O₈PNa[M+Na]⁺ 513.1151; found 513.1146.

3′-Benzoyl-2′-deoxy-L-thymidine-5′-(γ-(2-(pyrenesulfonyl)ethyl))triphosphate(Compound 30)

General procedure D with 170 mg (0.44 mmol) of compound 29, 248 mg (0.53mmol) of 2-(pyrenesulfonyl)ethyl]-pyrophosphate (compound 7) and 3 mL(4.40 mmol) of ZnCl₂ solution (1.5 M in anhydrous DMF) for 3 hoursstirring. After the reaction, the solution was precipitated by 150 mL ofether. Silica column chromatography with eluents (H₂O/(isopropanol-MeCN1:1) from 2% to 7% containing 1% diisopropylethylamine (DIPEA) to affordthe white solid compound 30; yield: 150 mg (46.7%); TLC (H₂O/isopropanol1:10 containing 2% DIPEA): Rf=0.35; 1H NMR (400 MHz, DMSO-d₆+D₂O (1:2))δ: 8.99 (brs, 1H), 8.65 (d, 1H, J=8 Hz), 8.32-8.21 (m, 5H), 8.15-8.09(m, 2H), 7.98-7.87 (m, 2H), 7.70 (s, 1H), 7.56-7.55 (m, 1H), 7.41 (s,1H), 6.22 (t, 1H, J=4 Hz), 5.10 (s, 1H), 4.35 (s, 2H), 4.22 (s, 1H),4.11 (s, 1H), 3.92 (t, 2H, J=4 Hz), 2.33 (s, 1H), 1.88 (s, 1H); ³¹P NMR(162 MHz, DMSO-d₆+D₂O (1:2)) δ −10.99 (brs, 1P), −11.26 (brs, 1P) −20.94(brs, 1P); HRMS (ESI-TOF) calcd. for C₃₅H₃₁N₂O₁₇P₃SNa [M−2H+Na]⁻899.0459; found 899.0454.

L-2′-deoxythymidine-5′-triphosphate (compound 31)

General procedure E with 20 mg (0.04 mmol) of compound 30, 10 mL of 33%NH₄OH_((aq)) for 15 hours stirring at room temperature. The product,compound 31, was afforded as a white solid; yield: 8 mg (83.2%); ¹H NMR(400 MHz, D₂O) δ 7.84 (s, 1H), 6.28 (t, 1H, J=6.0 Hz), 5.17 (s, 1H),4.54 (s, 1H), 4.15 (d, 1H, J=10.4 Hz), 2.83-2.77 (m, 1H), 2.49 (s, 1H),2.45 (s, 2H), 2.00 (s, 3H); ³¹P NMR (162 MHz, D₂O) δ −4.38 (d, J=16.2Hz), −10.65 (d, J=16.2 Hz), −19.74 (brs).

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. A composition comprising an organic moleculecomprising a phosphate moiety of formula (1a):

wherein: a) R_(1a) is selected from the group consisting of:

or b) the compound of formula (1a) is selected from the group consistingof:


2. The composition of claim 1, wherein R_(1a) is selected from the groupconsisting of:


3. The composition of claim 1, wherein the compound of formula (1a) isselected from the group consisting of:


4. A compound represented by formula (2):

wherein n is an integer from 1 to 10, and wherein R₂ is a support.
 5. Acompound represented by formula (3):

wherein p is an integer from 0 to 10, and R₃ is a support.
 6. A compoundrepresented by formula (4):

wherein q is an integer from 0 to 10, and r is an integer from 0 to 10.7. The compound of claim 6, wherein q is
 1. 8. The compound of claim 6,herein r is
 1. 9. The compound of claim 6, wherein the compound offormula (4) is


10. A method of synthesizing a nucleoside (n)phosphate, wherein (n) isselected from the group consisting of: di, tri, tetra, penta, and hexa,the method comprising a) contacting an activated nucleosidemonophosphate with a compound selected from the group consisting of: i)a compound represented by formula (1):

wherein: m is an integer from 0 to 5; n is an integer from 0 to 5; X isselected from the group consisting of O and CH₂; R₁₁ is selected fromthe group consisting of an aryl, a heteroaryL an alkyl, a cycloalkyl, analkenyl, an alkynyl, an alkyl-aryl, an aryl-alkyl, and a silyl groupwherein R₁₁ is optionally substituted; R₁₂ is hydrogen, null, asubstituted tetahydrofuranyl group, or an alkyl-substitutedtetahydrofuranyl group; and each occurrence of R₁₃ is independentlyhydrogen or null; ii) a compound represented by formula (1a);

wherein: R_(1a) is selected from the group consisting of an aryl, aheteroaryl, an alkyl, a cycloalkyl, an alkenyl, an alkynyl, analkyl-aryl, and an aryl-alkyl; and wherein R_(1a) is optionallysubstituted, iii) a compound represented by formula (1b):

wherein: R_(1b) is a hydroxy protecting group; iv) a compoundrepresented by formula (2):

wherein n is an integer from 1 to 10, and wherein R₂ is a support; v) acompound represented by formula (3):

wherein: p is an integer from 0 to 10, and R₃ is a support; and vi) acompound represented by formula (4):

wherein: q is an integer from 0 to 10, and r is an integer from 0 to 10,and an acid catalyst to catalyze the formation of a reactionintermediate, and b) contacting the reaction intermediate with a base toinduce formation of a nucleoside (n)phosphate.
 11. The method of claim10, wherein the acid catalyst is ZnCl₂.
 12. The method of claim 10,wherein the activated nucleoside monophosphate is represented by formula(5)

wherein R₅ is selected from the group of hydrogen, aryl, and heteroaryl;and R₆ is a nitrogenous base, wherein the nitrogenous base is a naturalnitrogenous base or artificial nitrogenous base.
 13. The method of claim10, wherein the activated nucleoside monophosphate is an activatedmonophosphate of a nucleoside selected from the group consisting ofadenosine, cytidine, guanosine, uridine, 2′-deoxyadenosine,2′-deoxycytidine, 2 ′-deoxyguanosine, thymidine, inosine,9-(β-D-arabinofuranosyl)adenine, 1-(β-D-arabinofuranosyl)cytosine,9-(β-D-arabinofuranosyl)guanine, 1-(β-D-arabinofuranosyl)uracil,9-(β-D-arabinofuranosyl)hypoxanthine, 1-(β-D-arabinofuranosyl)thymine,3′-azido-3′-deoxythymidine, 3′-azido-2′, 3′-dideoxyuridine,3′-dideoxycytidine, 3′-dideoxyadenosine, 3′-azido-2′,3′-dideoxyguanosine, 3′-azido-2′, 3′-dideoxyinosine, 3′-deoxythymidine,2′, 3′-dideoxyuridine, 2′, 3′-dideoxyinosine, 2′, 3′-dideoxyadenosine,2′, 3′-dideoxycytidine, 2′, 3′-dideoxyguanosine,9-(2,3-dideoxy-1-β-D-ribofuranosyl)-2, 6-diaminopurine, 3′-deoxy-2′,3′-didehydrothymidine, 2′, 3′-didehydro-2′, 3′-dideoxyuridine, 2′,3′-didehydro-2′, 3′-dideoxycytidine, 2′, 3′-didehydro-2′,3′-dideoxyadenosine, 2′, 3′-didehydro-2′, 3′-dideoxyguanosine, 2′,3′-didehydro-2′, 3′-dideoxyinosine, 3-deazaadenosine, 3-deazaguanosine,3-deazainosine, 7-deazaadenosine, 7-deazaguanosine, 7-deazainosine,6-azauridine, 6-azathymidine, 6-azacytidine, 5-azacytidine,9-(β-D-ribofuranosyl)-6-thiopurine,6-methylthio-9-(β-D-ribofuranosyl)purine,2-amino-9-(β-D-ribofuranosyl)-6-thiopurine,2-amino-6-methylthio-9-(β-D-ribofuranosyl)purine, 5-fluorocytidine,5-iodocytidine, 5-bromocytidine, 5-chiorocytidine, 5-fluorouridine,5-iodouridine, 5-bromouridine, 5-chlorouridine, 2′-C-methyladenosine,2′-C-methylcytidine, 2′-C-methylguanosine, 2′-C-methylinosine,2′-C-methyluridine, 2′-C-methylthymidine, 2′-deoxy-2′-fluoroadenosine,2′-deoxy-2′-fluorocytidine, 2′-deoxy-2′-fluoroguanosine,2′-deoxy-2′-fluorouridine, 2′-deoxy-2′-fluoroinosine,2′-α-fluorothymidine, 2′-deoxy-2′-fluoroarabinoadenosine,2′-deoxy-2′-fluoroarabinocytidine, 2′-deoxy-2′-fluoroarabinoguanosine,2′-deoxy-2′-fluoroarabinouridine, 2′-deoxy-2′-fluoroarabinoinosine,2′-β-fluorothymidine, 2′-O-methyladenosine, 2′-O-methylcytidine,methylguanosine, 2′-O-methylinosine, 2′-O-5-dimethyluridine,2′-C-ethynylcytidine, 2′-C-ethynylguanosine, 2′-C-ethynyluridine,2′-C-ethynylinosine, 2-C-ethynyl-5-methyluridine, 3′-C-ethynyladenosine,3′-C-ethynylcytidine, 3′-C-ethynylguanosine, 3′-C-ethynyluridine,3′-C-ethynylinosine, 3′-C-ethynyl-5-methyluridine, 3′-deoxyadenosine,3′-deoxycytidine, 3′-deoxyguanosine, 3′-deoxyuridine, 3′-deoxyinosine,4′-C-ethynyladenosine, 4′-C-ethynylcytidine, 4′-C-ethynylguanosine,4′-C-ethynyluridine, 4′-C-ethynylinosine, 4′-C-ethynyithymidine,4′-C-methyladenosine, 4′-C-methylcytidine, 4′-C-methylguanosine,4′-C-methyluridine, 4′-C-methylinosine, 4′-C-methylthymidine,2′C-methyl-7-deazaadenosine, 2′-C-methyl-7-deazaguanosine,2′-C-methyl-3-deazaadenosine, 2′-C-methyl-3-deazaguanosine,2′-O-methyl-7-deazaadenosine, 2′-O-methyl-7-deazaguanosine,2′-O-methyl-3-deazaadenosine, 2′-O-methyl-3-deazaguanosine,2′-C-methyl-6-azauridine, 2′-C-methyl-5-fluorouridine,2′-C-methyl-5-fluorocytidine, 2′-C-methyl-2-chloroadenosine,2′-deoxy-7-deazaadenosine, 2′-deoxy-3-deazaadenosine,2′-deoxy-7-deazaguanosine, 2′-deoxy-3-deazaguanosine,2′-deoxy-6-azauridine, 2′-deoxy-5-fluorouridine,2′-deoxy-5-fluorocytidine, 2′-deoxy-5-iodouridine,2′-deoxy-5-iodocytidine, 2′-deoxy-2-chloroadenosine,2′-deoxy-2-fluoroadenosine, 3′-deoxy-7-deazaadenosine,3′-deoxy-7-deazaguanosine, 3′-deoxy-3-deazaadenosine,3′-deoxy-3-deazaguanosine, 3′-deoxy-6-azauridine,3′-deoxy-5-fluorouridine, 3′-deoxy-5-iodouridine,3′-deoxy-5-fluorocytidine, 3′-deoxy-2-chloroadenosine, 2′,3′-dideoxy-7-deazaadenosine, 2′, 3′-dideoxy-7-deazaguanosine, 2′,3′-dideoxy-3-deazaadenosine, 2′, 3′-dideoxy-3-deazaguanosine, 2′,3′-dideoxy-6-azauridine, 2′, 3r-dideoxy-5-fluorouridine, 2′,3′-dideoxy-5-fluorouridine, 3′-dideoxy-5-iodocytidine, 2′,3?-dideoxy-2-chloroadenosine, 2′, 3′-dideoxy-β-L-cytidine, 2′,3′-dideoxy-β-L-adenosine, 2′, 3′-dideoxy-β-L-guanosine,3′-deoxy-β-L-thymidine, 2′, 3′-dideoxy-5-fluoro-β-L-cytidine,β-L-thyrnidine, 2′-deoxy-β-L-cytidine, 2′-deoxy-β-L-adenosine,2′-deoxy-β-L-guanosine, 2′-deoxy-β-L-inosine, β-L-cytidine,β-L-adenosine, β-L-guano sine, β-L-uridine, (3-L-inosine, 2′,3′-didehydro-2′, 3′-dideoxy-β-L-cytidine, 2′,3′-didehydro-3′-dideoxy-β-L-thymidine, 2′, 3′-didehydro-2′,3′-dideoxy-β-L-adenosine, 2′, 3′-didehydro-2′, 3′-dideoxy-β-L-guanosine,2′, 3′-didehydro-2′, 3′-dideoxy-β-L-5-fluorocytidine, 2′-deoxy-2′,2′-difluorocytidine, 9-(β-D-arabinofuranosyl)-2-fluoroadenine,2′-deoxy-2 ′(E)-fluoromethylenecytidine, 2′-deoxy-2′(Z)-fluoromethylenecytidine, (−) -2′, 3′-dideoxy-3′-thiacytidine,(+)-2′, 3′-dideoxy-3′-thiacytidine, 1-β-D-ribofuranosyl-1, 2,4-triazole-3-carboxamide, 1-β-L-ribofuranosyl-1, 2,4-triazole-3-carboxamide, 1-β-D-ribofuranosyl-1, 3-imidazolium-5-olate,1-β-L-ribofuranosyl-1, 3-imidazolium-5-olate,1-β-D-ribofuranosyl-5-ethynylimidazole-4-carboxamide,1-β-L-ribofuranosyl-5-ethynylimidazole-4-carboxamide,1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodouracil,1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodocytosine,1-(2-deoxy-2-fluoro-β-L-arabinofuranosyl)-5-methyluraeil,143-D-arabinofuranosyl-E-5-(2-bromovinyOuracil,E-5-(2-bromovinyl)-2′-deoxyuridine, 5-trifluoromethylthymidine,1-β-D-arabinoifuranosyl-5-propynyluracil,1-(2-deoxy-2-fluoro-1-β-D-arabinofuranosyl)-5-ethyluracil, 2′,3′-dideoxy-3′-fluoroguanosine, 3′-deoxy-3′-fluorothymidine, (±)-(1α, 2β,3α)-9-[2, 3-bis(hydroxymethyl)-1-cyclobutyl]adenine, (±)-(1α, 2β,3α)-9-[2, 3-bis(hydroxymethyl)-1-cyclobutyl]guanine, (±)-(1β2α,3β)-9-[2, 3-bis(hydroxymethyl)-1-cyclobutyl]guanine, (±)-(1β, 2α,3β)-9-[2, 3-bis(hydroxymethyl)-1-cyclobutyl]adenine, (1R, 3S,4R)-9-(3-hydroxy-4-hydroxymethylcyclopent-1-yl)guanine, (1S, 2R,4R)-9-(1-hydroxy-2-hydroxymethylcyclopent-4-yl)guanine, (2R,4R)-9-(2-hydroxymethyl-1, 3-dioxolan4-yl)-2, 6-diaminopurine, (2R,4R)-1-(2-hydroxymethyl-1, 3-dioxolan-4-yl)cytosine, (2R,4R)-9-(2-hydroxymethyl-1, 3-dioxolan-4-yl)guanine, (2R,4R)-1(2-hydroxymethyl-1, 3-dioxolan-4-yl)-5-fluorocytosine, (1R, 2S,4S)-9-(4-hydroxy-3-hydroxymethyl-2-methylenecyclopent-4-yl]guanine, and(1S, 3R,4S)-9-(3-hydroxy-4-hydroxymethyl-5-methylenecyclopent-1-yl]guanine. 14.A composition comprising at least one nucleoside (n)phosphatesynthesized according to the method of claim 10, wherein the methodcomprises: a) contacting an activated nucleoside monophosphate with acompound selected from the group consisting of: i) a compoundrepresented by formula 1:

wherein: R_(1a) is selected from the group consisting of an aryl, aheteroaryl, an alkyl, a cycloalkyl, an alkenyl, an alkynyl, analkyl-aryl and an aryl-alkyl; and wherein Ria is optionally substituted:ii) a compound represented by formula (1b):

wherein: R_(1b) is a hydroxy protecting group: iii) a compoundrepresented by formula (2):

wherein n is an integer from 1 to 10, and wherein R₂ is a support, iv) acompound represented by formula (3):

wherein: p is an integer from 0 to 10, and R₃ is a support; and v) acompound represented by formula (4):

wherein: q is an integer from 0 to 10, and r is an integer from 0 to 10,and an acid catalyst to catalyze the formation of a reactionintermediate, and b) contacting the reaction intermediate with a base toinduce formation of a nucleoside (n)phosphate.
 15. The composition ofclaim 14, wherein the composition comprises at least four nucleosidetriphosphates.
 16. The composition of claim 15, wherein at least fournucleoside triphosphates are deoxyadenosine triphosphate (dATP),deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP),and deoxythymidine triphosphate (dTTP).
 17. The composition of claim 14,wherein the composition is a pharmaceutical composition.
 18. A method oftreating a disease or disorder in a subject in need thereof, the methodcomprising administering to the subject a composition of claim
 17. 19. Akit comprising at least one nucleoside (n)phosphate synthesizedaccording to the method of claim 10; wherein the method comprises: a)contacting an activated nucleoside monophosphate with a compoundselected from the group consisting of: i) a compound represented bformula (1a):

wherein: R_(1a) is selected from the group consisting of an aryl, aheteroaryl, an alkyl, a cycloalkyl, an alkenyl, an alkynyl, analkyl-aryl, and an aryl-alkyl; and wherein Ria is optionallysubstituted; ii) a compound represented by formula (1b):

wherein: R_(1b) is a hydroxy protecting group; iii) a compoundrepresented by formula (2):

wherein n is an integer from 1 to 10, and wherein R₂ is a support; iv) acompound represented by formula (3):

wherein: p is an integer from 0 to 10, and R₃ is a support; and v) acompound represented by formula (4):

wherein: q is an integer from 0 to 10, and r is an integer from 0 to 10,and an acid catalyst to catalyze the formation of a reactionintermediate, and b) contacting the reaction intermediate with a base toinduce formation of a nucleoside (n)phosphate.
 20. The kit of claim 19,wherein the composition comprises at least four nucleosidetriphosphates.
 21. The composition of claim 20, wherein at least fournucleoside triphosphates are deoxyadenosine triphosphate (dATP),deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP),and deoxythymidine triphosphate (dTTP).